Identification of patients in need of PD-l1 inhibitor cotherapy

Abstract

The present invention relates to means and methods for determining whether a patient is in need of a PD-L1 inhibitor cotherapy. A patient is determined to be in need of the PD-L1 inhibitor cotherapy if a low or absent ER expression level and an expression level of programmed death ligand 1 (PD-L1) that is increased in comparison to a control is measured in vitro in a sample from the patient. The patient is undergoing therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway (like Trastuzumab) and a chemotherapeutic agent (like dodetaxel) or such a therapy is contemplated for the patient. Also provided herein are means and methods for treating a cancer in a cancer patient for whom therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway (like Trastuzumab) and a chemotherapeutic agent (like dodetaxel) is contemplated, wherein the patient is to receive PD-L1 inhibitor cotherapy.

Description

CROSS-REFERENCE RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent application Ser. No. 17 / 744,271, filed on May 13, 2022 (Pending), which is a continuation of U.S. patent application Ser. No. 17 / 674,615 filed on Feb. 17, 2022 (Abandoned), which is a continuation of U.S. patent application Ser. No. 17 / 483,396 filed on Sep. 23, 2021 (Abandoned), which is a continuation of U.S. patent application Ser. No. 17 / 323,120 filed on May 18, 2021 (Abandoned), which is a continuation of U.S. patent application Ser. No. 16 / 814,688 filed on Mar. 10, 2020 (Abandoned), which is a continuation of U.S. patent application Ser. No. 15 / 815,384, filed Nov. 16, 2017 (Abandoned), which is a continuation of U.S. patent application Ser. No. 14 / 720,643, filed May 22, 2015 (Abandoned), which is a continuation of International Patent Application No. PCT / EP2013 / 075162, filed Nov. 29, 2013 (Expired), which claims priority to European Patent Application No. 12195182.6, filed Nov. 30, 2012 (Abandoned) and European Patent Application No. 12196177.5, filed Dec. 7, 2012 (Abandoned), the disclosures of each of which are incorporated by reference herein in their entireties.SEQUENCE LISTING

[0002] This application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Feb. 26, 2025 is named P31922-US-7_Sequence Listing.xml and is 625,775 bytes in size.BACKGROUND

[0003] The present invention relates to means and methods for determining whether a patient is in need of a PD-L1 inhibitor cotherapy. A patient is determined to be in need of the PD-L1 inhibitor cotherapy if a low or absent ER expression level and an expression level of programmed death ligand 1 (PD-L1) that is increased in comparison to a control is measured in vitro in a sample from the patient. The patient is undergoing therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway (like Trastuzumab) and a chemotherapeutic agent (like dodetaxel) or such a therapy is contemplated for the patient. Also provided herein are means and methods for treating a cancer in a cancer patient for whom therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway (like Trastuzumab) and a chemotherapeutic agent (like dodetaxel) is contemplated, wherein the patient is to receive PD-L1 inhibitor cotherapy.

[0004] The HER family of receptor tyrosine kinases are important mediators of cell growth, differentiation and survival. The receptor family includes four distinct members including epidermal growth factor receptor (EGFR, ErbB1, or HER1), HER2 (ErbB2 or p185neu), HER3 (ErbB3) and HER4 (ErbB4 or tyro2).

[0005] EGFR, encoded by the erbB1 gene, has been causally implicated in human malignancy. In particular, increased expression of EGFR has been observed in breast, bladder, lung, head, neck and stomach cancer as well as glioblastomas. Increased EGFR receptor expression is often associated with increased production of the EGFR ligand, transforming growth factor alpha (TGF-α), by the same tumor cells resulting in receptor activation by an autocrine stimulatory pathway. Baselga and Mendelsohn Pharmac. Ther. 64:127-154 (1994). Monoclonal antibodies directed against the EGFR or its ligands, TGF-α and EGF, have been evaluated as therapeutic agents in the treatment of such malignancies. See, e.g., Baselga and Mendelsohn., supra; Masui et al. Cancer Research 44:1002-1007 (1984); and Wu et al. J. Clin. Invest. 95:1897-1905 (1995).

[0006] The second member of the HER family, p185neu, was originally identified as the product of the transforming gene from neuroblastomas of chemically treated rats. The activated form of the neu proto-oncogene results from a point mutation (valine to glutamic acid) in the transmembrane region of the encoded protein. Amplification of the human homolog of neu is observed in breast and ovarian cancers and correlates with a poor prognosis (Slamon et al., Science, 235:177-182 (1987); Slamon et al., Science, 244:707-712 (1989); and U.S. Pat. No. 4,968,603). To date, no point mutation analogous to that in the neu proto-oncogene has been reported for human tumors. Overexpression of HER2 (frequently but not uniformly due to gene amplification) has also been observed in other carcinomas including carcinomas of the stomach, endometrium, salivary gland, lung, kidney, colon, thyroid, pancreas and bladder. See, among others, King et al., Science, 229:974 (1985); Yokota et al., Lancet: 1:765-767 (1986); Fukushige et al., Mol Cell Biol., 6:955-958 (1986); Guerin et al., Oncogene Res., 3:21-31 (1988); Cohen et al., Oncogene, 4:81-88 (1989); Yonemura et al., Cancer Res., 51:1034 (1991); Borst et al., Gynecol. Oncol., 38:364 (1990); Weiner et al., Cancer Res., 50:421-425 (1990); Kern et al., Cancer Res., 50:5184 (1990); Park et al., Cancer Res., 49:6605 (1989); Zhau et al., Mol. Carcinog., 3:254-257 (1990); Aasland et al. Br. J. Cancer 57:358-363 (1988); Williams et al. Pathobiology 59:46-52 (1991); and McCann et al., Cancer, 65:88-92 (1990). HER2 may be overexpressed in prostate cancer (Gu et al. Cancer Lett. 99:185-9 (1996); Ross et al. Hum. Pathol. 28:827-33 (1997); Ross et al. Cancer 79:2162-70 (1997); and Sadasivan et al. J. Urol. 150:126-31 (1993)).

[0007] Antibodies directed against the rat p185neu and human HER2 protein products have been described. Drebin and colleagues have raised antibodies against the rat neu gene product, p185neu. See, for example, Drebin et al., Cell 41:695-706 (1985); Myers et al., Meth. Enzym. 198:277-290 (1991); and WO94 / 22478. Drebin et al. Oncogene 2:273-277 (1988) report that mixtures of antibodies reactive with two distinct regions of p185neu result in synergistic anti-tumor effects on neu-transformed NIH-3T3 cells implanted into nude mice. See also U.S. Pat. No. 5,824,311 issued Oct. 20, 1998.

[0008] Hudziak et al., Mol. Cell. Biol. 9(3):1165-1172 (1989) describe the generation of a panel of HER2 antibodies which were characterized using the human breast tumor cell line SK-BR-3. Relative cell proliferation of the SK-BR-3 cells following exposure to the antibodies was determined by crystal violet staining of the monolayers after 72 hours. Using this assay, maximum inhibition was obtained with the antibody called 4D5 which inhibited cellular proliferation by 56%. Other antibodies in the panel reduced cellular proliferation to a lesser extent in this assay. The antibody 4D5 was further found to sensitize HER2-overexpressing breast tumor cell lines to the cytotoxic effects of TNF-α. See also U.S. Pat. No. 5,677,171 issued Oct. 14, 1997. The HER2 antibodies discussed in Hudziak et al. are further characterized in Fendly et al. Cancer Research 50:1550-1558 (1990); Kotts et al. In Vitro 26(3):59A (1990); Sarup et al. Growth Regulation 1:72-82 (1991); Shepard et al. J. Clin. Immunol. 11(3):117-127 (1991); Kumar et al. Mol. Cell. Biol. 11(2):979-986 (1991); Lewis et al. Cancer Immunol. Immunother. 37:255-263 (1993); Pietras et al. Oncogene 9:1829-1838 (1994); Vitetta et al. Cancer Research 54:5301-5309 (1994); Sliwkowski et al. J. Biol. Chem. 269(20):14661-14665 (1994); Scott et al. J. Biol. Chem. 266:14300-5 (1991); D'souza et al. Proc. Natl. Acad. Sci. 91:7202-7206 (1994); Lewis et al. Cancer Research 56:1457-1465 (1996); and Schaefer et al. Oncogene 15:1385-1394 (1997).

[0009] A recombinant humanized version of the murine HER2 antibody 4D5 (huMAb4D5-8, rhuMAb HER2, Trastuzumab or Herceptin™; U.S. Pat. No. 5,821,337) is clinically active in patients with HER2-overexpressing metastatic breast cancers that have received extensive prior anti-cancer therapy (Baselga et al., J. Clin. Oncol. 14:737-744 (1996)). Trastuzumab received marketing approval from the Food and Drug Administration Sep. 25, 1998 for the treatment of patients with metastatic breast cancer whose tumors overexpress the HER2 protein.

[0010] Humanized anti-ErbB2 antibodies include huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 (HERCEPTIN®) as described in Table 3 of U.S. Pat. No. 5,821,337 expressly incorporated herein by reference; humanized 520C9 (WO 93 / 21319) and humanized 2C4 antibodies as described in WO 01 / 000245 expressly incorporated herein by reference.

[0011] Pertuzumab (see e.g. WO 01 / 000245) is the first of a new class of agents known as HER dimerization inhibitors (HDIs). Pertuzumab binds to HER2 at its dimerization domain, thereby inhibiting its ability to form active dimer receptor complexes and thus blocking the downstream signal cascade that ultimately results in cell growth and division (see Franklin, M. C., Cancer Cell 5 (2004) 317-328). Pertuzumab is a fully humanized recombinant monoclonal antibody directed against the extracellular domain of HER2. Binding of Pertuzumab to the HER2 on human epithelial cells prevents HER2 from forming complexes with other members of the HER family (including EGFR, HER3, HER4) and probably also HER2 homodimerization. By blocking complex formation, Pertuzumab prevents the growth stimulatory effects and cell survival signals activated by ligands of HER1, HER3 and HER4 (e.g. EGF, TGFalpha, amphiregulin, and the heregulins). Another name for Pertuzumab is 2C4. Pertuzumab is a fully humanized recombinant monoclonal antibody based on the human IgG1(K) framework sequences. The structure of Pertuzumab consists of two heavy chains (449 residues) and two light chains (214 residues). Compared to Trastuzumab (Herceptin®), Pertuzumab has 12 amino acid differences in the light chain and 29 amino acid differences in the IgG1 heavy chain.

[0012] Other HER2 antibodies with various properties have been described in Tagliabue et al. Int. J. Cancer 47:933-937 (1991); McKenzie et al. Oncogene 4:543-548 (1989); Maier et al. Cancer Res. 51:5361-5369 (1991); Bacus et al. Molecular Carcinogenesis 3:350-362 (1990); Stancovski et al. PNAS (USA) 88:8691-8695 (1991); Bacus et al. Cancer Research 52:2580-2589 (1992); Xu et al. Int. J. Cancer 53:401-408 (1993); WO94 / 00136; Kasprzyk et al. Cancer Research 52:2771-2776 (1992); Hancock et al. Cancer Res. 51:4575-4580 (1991); Shawver et al. Cancer Res. 54:1367-1373 (1994); Arteaga et al. Cancer Res. 54:3758-3765 (1994); Harwerth et al. J. Biol. Chem. 267:15160-15167 (1992); U.S. Pat. No. 5,783,186; and Klapper et al. Oncogene 14:2099-2109 (1997).

[0013] Homology screening has resulted in the identification of two other HER receptor family members; HER3 (U.S. Pat. Nos. 5,183,884 and 5,480,968 as well as Kraus et al. PNAS (USA) 86:9193-9197 (1989)) and HER4 (EP Pat. Appln. No 599,274; Plowman et al., Proc. Natl. Acad. Sci. USA, 90:1746-1750 (1993); and Plowman et al., Nature, 366:473-475 (1993)). Both of these receptors display increased expression on at least some breast cancer cell lines.

[0014] The HER receptors are generally found in various combinations in cells and heterodimerization is thought to increase the diversity of cellular responses to a variety of HER ligands (Earp et al. Breast Cancer Research and Treatment 35: 115-132 (1995)). EGFR is bound by six different ligands; epidermal growth factor (EGF), transforming growth factor alpha (TGF-α), amphiregulin, heparin binding epidermal growth factor (HB-EGF), betacellulin and epiregulin (Groenen et al. Growth Factors 11:235-257 (1994)). A family of heregulin proteins resulting from alternative splicing of a single gene are ligands for HER3 and HER4. The heregulin family includes alpha, beta and gamma heregulins (Holmes et al., Science, 256:1205-1210 (1992); U.S. Pat. No. 5,641,869; and Schaefer et al. Oncogene 15:1385-1394 (1997)); neu differentiation factors (NDFs), glial growth factors (GGFs); acetylcholine receptor inducing activity (ARIA); and sensory and motor neuron derived factor (SMDF). For a review, see Groenen et al. Growth Factors 11:235-257 (1994); Lemke, G. Molec. &Cell. Neurosci. 7:247-262 (1996) and Lee et al. Pharm. Rev. 47:51-85 (1995). Recently three additional HER ligands were identified; neuregulin-2 (NRG-2) which is reported to bind either HER3 or HER4 (Chang et al. Nature 387 509-512 (1997); and Carraway et al Nature 387:512-516 (1997)); neuregulin-3 which binds HER4 (Zhang et al. PNAS (USA) 94(18):9562-7 (1997)); and neuregulin-4 which binds HER4 (Harari et al. Oncogene 18:2681-89 (1999)) HB-EGF, betacellulin and epiregulin also bind to HER4.

[0015] While EGF and TGFα do not bind HER2, EGF stimulates EGFR and HER2 to form a heterodimer, which activates EGFR and results in transphosphorylation of HER2 in the heterodimer. Dimerization and / or transphosphorylation appears to activate the HER2 tyrosine kinase. See Earp et al., supra. Likewise, when HER3 is co-expressed with HER2, an active signaling complex is formed and antibodies directed against HER2 are capable of disrupting this complex (Sliwkowski et al., J. Biol. Chem., 269(20):14661-14665 (1994)). Additionally, the affinity of HER3 for heregulin (HRG) is increased to a higher affinity state when co-expressed with HER2. See also, Levi et al., Journal of Neuroscience 15: 1329-1340 (1995); Morrissey et al., Proc. Natl. Acad. Sci. USA 92: 1431-1435 (1995); and Lewis et al., Cancer Res., 56:1457-1465 (1996) with respect to the HER2-HER3 protein complex. HER4, like HER3, forms an active signaling complex with HER2 (Carraway and Cantley, Cell 78:5-8 (1994)).

[0016] Also, antibody variant compositions are described in the art. U.S. Pat. No. 6,339,142 describes a HER2 antibody composition comprising a mixture of anti-HER2 antibody and one or more acidic variants thereof, wherein the amount of the acidic variant(s) is less than about 25%. Trastuzumab is the exemplified HER2 antibody. Reid et al. Poster presented at Well Characterized Biotech Pharmaceuticals conference (January, 2003) “Effects of Cell Culture Process Changes on Humanized Antibody Characteristics” describes an unnamed, humanized IgG1 antibody composition with N-terminal heterogeneities due to combinations of VHS signal peptide, N-terminal glutamine, and pyroglutamic acid on the heavy chain thereof. Harris et al. “The Ideal Chromatographic Antibody Characterization Method” talk presented at the IBC Antibody Production Conference (February, 2002) reports a VHS extension on the heavy chain of E25, a humanized anti-IgE antibody. Rouse et al. Poster presented at WCBP “Glycoprotein Characterization by High Resolution Mass Spectrometry and Its Application to Biopharmaceutical Development” (Jan. 6-9, 2004) describes a monoclonal antibody composition with N-terminal heterogeneity resulting from AHS or HS signal peptide residues on the light chain thereof. In a presentation at IBC Meeting (September, 2000) “Strategic Use of Comparability Studies and Assays for Well Characterized Biologicals,” Jill Porter discussed a late-eluting form of ZENAPAX™ with three extra amino acid residues on the heavy chain thereof. US2006 / 0018899 describes a composition comprising a main species pertuzumab antibody and an amino-terminal leader extension variant, as well as other variant forms of the pertuzumab antibody.

[0017] Patent publications related to HER antibodies include: U.S. Pat. Nos. 5,677,171, 5,720,937, 5,720,954, 5,725,856, 5,770,195, 5,772,997, 6,165,464, 6,387,371, 6,399,063, US2002 / 0192211A1, U.S. Pat. Nos. 6,015,567, 6,333,169, 4,968,603, 5,821,337, 6,054,297, 6,407,213, 6,719,971, 6,800,738, US2004 / 0236078A1, U.S. Pat. Nos. 5,648,237, 6,267,958, 6,685,940, 6,821,515, WO98 / 17797, U.S. Pat. Nos. 6,127,526, 6,333,398, 6,797,814, 6,339,142, 6,417,335, 6,489,447, WO99 / 31140, US2003 / 0147884A1, US2003 / 0170234A1, US2005 / 0002928A1, U.S. Pat. No. 6,573,043, US2003 / 0152987A1, WO99 / 48527, US2002 / 0141993A1, WO01 / 00245, US2003 / 0086924, US2004 / 0013667A1, WO00 / 69460, WO01 / 00238, WO01 / 15730, U.S. Pat. Nos. 6,627,196B1, 6,632,979B1, WO01 / 00244, US2002 / 0090662A1, WO01 / 89566, US2002 / 0064785, US2003 / 0134344, WO 04 / 24866, US2004 / 0082047, US2003 / 0175845A1, WO03 / 087131, US2003 / 0228663, WO2004 / 008099A2, US2004 / 0106161, WO2004 / 048525, US2004 / 0258685A1, U.S. Pat. Nos. 5,985,553, 5,747,261, 4,935,341, 5,401,638, 5,604,107, WO 87 / 07646, WO 89 / 10412, WO 91 / 05264, EP 412,116 B1, EP 494,135 B1, U.S. Pat. No. 5,824,311, EP 444,181 B1, EP 1,006,194 A2, US 2002 / 0155527A1, WO 91 / 02062, U.S. Pat. Nos. 5,571,894, 5,939,531, EP 502,812 B1, WO 93 / 03741, EP 554,441 B1, EP 656,367 A1, U.S. Pat. Nos. 5,288,477, 5,514,554, 5,587,458, WO 93 / 12220, WO 93 / 16185, U.S. Pat. No. 5,877,305, WO 93 / 21319, WO 93 / 21232, U.S. Pat. No. 5,856,089, WO 94 / 22478, U.S. Pat. Nos. 5,910,486, 6,028,059, WO 96 / 07321, U.S. Pat. Nos. 5,804,396, 5,846,749, EP 711,565, WO 96 / 16673, U.S. Pat. Nos. 5,783,404, 5,977,322, 6,512,097, WO 97 / 00271, U.S. Pat. Nos. 6,270,765, 6,395,272, 5,837,243, WO 96 / 40789, U.S. Pat. Nos. 5,783,186, 6,458,356, WO 97 / 20858, WO 97 / 38731, U.S. Pat. Nos. 6,214,388, 5,925,519, WO 98 / 02463, U.S. Pat. No. 5,922,845, WO 98 / 18489, WO 98 / 33914, U.S. Pat. No. 5,994,071, WO 98 / 45479, U.S. Pat. No. 6,358,682 B1, US 2003 / 0059790, WO 99 / 55367, WO 01 / 20033, US 2002 / 0076695 A1, WO 00 / 78347, WO 01 / 09187, WO 01 / 21192, WO 01 / 32155, WO 01 / 53354, WO 01 / 56604, WO 01 / 76630, WO02 / 05791, WO 02 / 11677, U.S. Pat. No. 6,582,919, US2002 / 0192652A1, US 2003 / 0211530A1, WO 02 / 44413, US 2002 / 0142328, U.S. Pat. No. 6,602,670 B2, WO 02 / 45653, WO 02 / 055106, US 2003 / 0152572, US 2003 / 0165840, WO 02 / 087619, WO 03 / 006509, WO03 / 012072, WO 03 / 028638, US 2003 / 0068318, WO 03 / 041736, EP 1,357,132, US 2003 / 0202973, US 2004 / 0138160, U.S. Pat. Nos. 5,705,157, 6,123,939, EP 616,812 B1, US 2003 / 0103973, US 2003 / 0108545, U.S. Pat. No. 6,403,630 B1, WO 00 / 61145, WO 00 / 61185, U.S. Pat. No. 6,333,348 B1, WO 01 / 05425, WO 01 / 64246, US 2003 / 0022918, US 2002 / 0051785 A1, U.S. Pat. No. 6,767,541, WO 01 / 76586, US 2003 / 0144252, WO 01 / 87336, US 2002 / 0031515 A1, WO 01 / 87334, WO 02 / 05791, WO 02 / 09754, US 2003 / 0157097, US 2002 / 0076408, WO 02 / 055106, WO 02 / 070008, WO 02 / 089842 and WO 03 / 86467.

[0018] Patients treated with the HER2 antibody Trastuzumab / Herceptin™ are selected for therapy based on HER2 protein overexpression / gene amplification; see, for example, WO99 / 31140 (Paton et al.), US2003 / 0170234A1 (Hellmann, S.), and US2003 / 0147884 (Paton et al.); as well as WO01 / 89566, US2002 / 0064785, and US2003 / 0134344 (Mass et al.). See, also, US2003 / 0152987, Cohen et al., concerning immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) for detecting HER2 overexpression and amplification. WO2004 / 053497 and US2004 / 024815A1 (Bacus et al.), as well as US 2003 / 0190689 (Crosby and Smith), refer to determining or predicting response to Trastuzumab therapy. US2004 / 013297A1 (Bacus et al.) concerns determining or predicting response to ABX0303 EGFR antibody therapy. WO2004 / 000094 (Bacus et al.) is directed to determining response to GW572016, a small molecule, EGFR-HER2 tyrosine kinase inhibitor. WO2004 / 063709, Amler et al., refers to biomarkers and methods for determining sensitivity to EGFR inhibitor, erlotinib HCl. US2004 / 0209290, Cobleigh et al., concerns gene expression markers for breast cancer prognosis.DETAILED DESCRIPTION

[0019] Patients to be treated with a HER2 dimerization inhibitor (like pertuzumab as described herein above in more detail) can be selected for therapy based on HER activation or dimerization. Patent publications concerning pertuzumab and selection of patients for therapy therewith include: WO01 / 00245 (Adams et al.); US2003 / 0086924 (Sliwkowski, M.); US2004 / 0013667A1 (Sliwkowski, M.); as well as WO2004 / 008099A2, and US2004 / 0106161 (Bossenmaier et al.).

[0020] Herceptin™ / Trastuzumab is indicated in the art for the treatment of patients with metastatic breast cancer whose tumors overexpress HER2 protein or have HER 2 gene amplification:

[0021] a) As monotherapy for the treatment of those patients who have received at least two chemotherapy regimens for their metastatic disease. Prior chemotherapy must have included at least an anthracycline and a taxane unless patients are unsuitable for these treatments. Hormone receptor positive patients must also have received hormonal therapy, unless patients are unsuitable for these treatments,

[0022] b) In combination with paclitaxel for the treatment of those patients who have not received chemotherapy for their metastatic disease and for whom an anthracycline is not suitable and

[0023] c) In combination with docetaxel for the treatment of those patients who have not received chemotherapy for their metastatic disease.

[0024] Herceptin™ / Trastuzumab can also be used as adjuvant treatment in early breast cancer. Herceptin™ / Trastuzumab is also approved for the treatment of patients with HER2-positive early breast cancer following surgery, chemotherapy (neoadjuvant (i.e. before surgery) or adjuvant), and radiotherapy (if applicable). In addition, Herceptin in combination with capecitabine or 5-fluorouracil and cisplatin is indicated for the treatment of patients with HER2 positive locally advance or metastatic adenocarcinoma of the stomach or gastroesophageal junction who have not received prior anti-cancer treatment for their metastatic disease. The efficacy and safety of neoadjuvant pertuzumab and trastuzumab therapy has been assessed in a phase 2 trial (NEOSPHERE); Gianni (2012) Lancet Oncol 13, 25-32.

[0025] In the art, the treatment of breast cancer patients with Herceptin™ / Trastuzumab is, for example, recommended and routine for patients having HER2-positive cancer. HER2-positive cancer is present if a high HER2 (protein) expression level detected by immunohistochemical methods (e.g. HER2 (+++)) or HER2 gene amplification detected by in-situ-hybridization (e.g. ISH positive, like a HER2 gene copy number higher than 4 copies of the HER2 gene per tumor cell or ratio of ≥2.0 for the number of HER2 gene copies to the number of signals for CEP17.) or both is found in samples obtained from the patients such as breast tissue biopsies or breast tissue resections or in tissue derived from metastatic sites.

[0026] WO 2011 / 109789, WO 2011 / 066342, WO 2009 / 089149 and WO2006 / 133396 disclose the therapeutic use of PD-L1 inhibitors. Moreover, WO 2010 / 077634 discloses anti-PD-L1 antibodies and their therapeutic use.

[0027] The present invention relates to a method of determining the need of a cancer patient for a PD-L1 inhibitor cotherapy, (i) wherein therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated for the patient or (ii) wherein the patient is undergoing therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent, said method comprising the steps of

[0028] a) measuring in vitro in a sample from said patient the expression level of Estrogen receptor (ER) and of programmed death ligand 1 (PD-L1),

[0029] b) determining a patient as being in need of a PD-L1 inhibitor cotherapy if a low or absent ER expression level and an expression level of programmed death ligand 1 (PD-L1) that is increased in comparison to a control is measured in step (a).

[0030] Accordingly, the present invention provides a method for determining a cancer patient's need for PD-L1 modulator cotherapy in combination with a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent, the method comprising the steps of

[0031] testing a tumor sample of a patient for whom therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated or who is undergoing said therapy;

[0032] determining the expression level of Estrogen receptor (ER) and of programmed death ligand 1 (PD-L1) in said tumor sample,

[0033] whereby a low or absent ER expression level and an expression level of programmed death ligand 1 (PD-L1) that is increased in comparison to the control is indicative of a successful use of PD-L1 modulator cotherapy in said patient.

[0034] As demonstrated in the appended example, it has been surprisingly found in this invention that Estrogen receptor (ER) negative (ER(−)) cancer patients (cancer patients with a low or even absent ER expression level) undergoing therapy with a modulator of the HER2 / neu (ErbB2) signaling pathway (like Herceptin™ / Trastuzumab) and a chemotherapeutic agent (like dodetaxel / Taxotere®) show a significantly worse pathological complete response (pCR) to the therapy compared to Estrogen receptor (ER) positive (ER(+)) cancer patients, if the expression level of programmed death ligand 1 (PD-L1) is increased in a sample of the ER negative (ER(−)) cancer patients as compared to a control. The terms “programmed death ligand 1”, “CD274” and “PD-L1” are used interchangeably herein. The ER negative (ER(−)) cancer patients with increased expression level of programmed death ligand 1 (PD-L1) as compared to a control will therefore benefit from additional cotherapy with a PD-L1 inhibitor. It is expected that the pathological complete response rate (pCR) in this patient group will increase, if these patients receive cotherapy with a PD-L1 inhibitor in addition to therapy with a modulator of the HER2 / neu (ErbB2) signaling pathway (like Herceptin™ / Trastuzumab) and a chemotherapeutic agent (like dodetaxel / Taxotere®). In other words, the ER negative (ER(−)) cancer patients are to receive a programmed death ligand 1 (PD-L1) inhibitor in addition to a modulator of the HER2 / neu (ErbB2) signaling pathway (like Trastuzumab) and a chemotherapeutic agent (like dodetaxel / Taxotere®), if the expression level of programmed death ligand 1 (PD-L1) is increased in a sample from the patient in comparison to a control. In the following, ER negative cancer patients or (biological / tumor) samples derived from ER negative cancer patients are denoted herein as “ER(−)”. Likewise ER positive cancer patients or (biological / tumor) samples derived from ER positive cancer patients are denoted herein as “ER(+)”.

[0035] In accordance with the above, the present invention relates to a method of treating a cancer in a cancer patient for whom therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated, the method comprising selecting a cancer patient whose cancer is determined to have a low or absent ER expression level and to have an increased expression level of programmed death ligand 1 (PD-L1) in comparison to a control, and administering to the patient an effective amount of a modulator of the HER2 / neu (ErbB2) signaling pathway, of a chemotherapeutic agent and of a programmed death ligand 1 (PD-L1) inhibitor. Likewise, the present invention relates to a method of treating a cancer in a cancer patient who is undergoing therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent, the method comprising selecting a cancer patient whose cancer is determined to have a low or absent ER expression level and to have an increased expression level of programmed death ligand 1 (PD-L1) in comparison to a control, and administering to the patient an effective amount of a programmed death ligand 1 (PD-L1) inhibitor. Herein contemplated is, accordingly, a pharmaceutical composition comprising a modulator of the HER2 / neu (ErbB2) signaling pathway, and an inhibitor of programmed death ligand 1 (PD-L1) for use in the treatment of cancer, whereby said cancer is determined to have a low or absent ER expression level and to have an increased expression level of programmed death ligand 1 (PD-L1) in comparison to a control.

[0036] In accordance with the above, the herein provided method for determining the need of a cancer patient for a PD-L1 inhibitor cotherapy, may comprise an additional step prior to step a), wherein said step is or comprises obtaining a sample from said cancer patient. Accordingly, the present invention provides a method of determining the need of a cancer patient for a PD-L1 inhibitor cotherapy, (i) wherein therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated for the patient or (ii) wherein the patient is undergoing therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent, said method comprising a step of obtaining a sample from said cancer patient, the method further comprising the steps

[0037] a) measuring in vitro in a sample from said patient the expression level of Estrogen receptor (ER) and of programmed death ligand 1 (PD-L1),

[0038] b) determining a patient as being in need of a PD-L1 inhibitor cotherapy if a low or absent ER expression level and an expression level of programmed death ligand 1 (PD-L1) that is increased in comparison to a control is measured in step (a).

[0039] Furthermore, it has been found herein and is demonstrated in the appended example, that a patient's need of PD-L1 inhibitor cotherapy can be determined even more reliably, if the expression level of interferon-gamma (IFNγ) is measured in the sample of the patient in addition to the expression level of programmed death ligand 1 (PD-L1). It is shown herein that patients with low or absent ER expression have a significantly worse pathologic complete response to therapy with a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent, if the expression level of programmed death ligand 1 (PD-L1) is increased and if the expression level of interferon-gamma (IFNγ) is decreased.

[0040] Accordingly, the methods provided herein preferably further comprise measuring the expression level of interferon-gamma (IFNγ) in the sample from the patient, whereby a patient is determined to be in need of a PD-L1 inhibitor cotherapy, if the expression level of interferon-gamma (IFNγ) is decreased in comparison to a control. In accordance with the above, the present invention relates in a preferred aspect to a method of determining the need of a cancer patient for a PD-L1 inhibitor cotherapy, (i) wherein therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated for the patient or (ii) wherein the patient is undergoing therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent, said method comprising the steps of

[0041] a) measuring in vitro in a sample from said patient the expression level of Estrogen receptor (ER), the expression level of programmed death ligand 1 (PD-L1), and the expression level of interferon-gamma (IFNγ)

[0042] b) determining a patient as being in need of a PD-L1 inhibitor cotherapy if a low or absent ER expression level, an expression level of programmed death ligand 1 (PD-L1) that is increased in comparison to a control, and an expression level of interferon-gamma (IFNγ) that is decreased in comparison to a control is measured in step (a).

[0043] Accordingly, an expression level of interferon-gamma (IFNγ) that is decreased in comparison to a control is indicative of a successful use of PD-L1 inhibitor cotherapy in said patient. The herein provided pharmaceutical composition is, in accordance with the above, for use in the treatment of cancer, whereby said cancer is determined to have a low or absent ER expression level, the cancer is determined to have an increased expression level of programmed death ligand 1 (PD-L1) in comparison to a control and the cancer is determined to have a decreased expression level of interferon-gamma (IFNγ) in comparison to the control. Accordingly, a pharmaceutical composition is provided herein comprising a modulator of the HER2 / neu (ErbB2) signaling pathway, and an inhibitor of programmed death ligand 1 (PD-L1) for use in the treatment of cancer, whereby said cancer is determined to have a low or absent ER expression level and to have an increased expression level of programmed death ligand 1 (PD-L1) in comparison to a control and to have a decreased expression level of interferon-gamma (IFNγ) in comparison to the control.

[0044] The term “cancer patient” as used herein refers to a patient that is suspected to suffer from cancer, suffering from cancer or being prone to suffer from cancer. The cancer to be treated in accordance with the present invention can be a solid cancer, such as breast cancer or gastric cancer. Further, the cancer may be ovarian cancer or colorectal cancer. The cancer is preferably a “HER2-positive” cancer.

[0045] Preferably, the cancer is breast cancer, like early breast cancer. The breast cancer may be early stage breast cancer or metastatic breast cancer. Accordingly, the cancer patient (to be treated) is suspected to suffer from solid cancer, is suffering from solid cancer or is being prone to suffer from solid cancer, whereby the solid cancer can be breast cancer or gastric cancer. Preferably, the cancer is breast cancer, like early stage breast cancer. The patient is preferably a human.

[0046] As mentioned above, the expression level of Estrogen receptor (ER) and of programmed death ligand (PD-L1), and optionally of interferon-gamma (IFN-γ) can be measured in vitro in a sample from the patient. Preferably, the herein provided methods comprise measuring of interferon-gamma (IFN-γ) in vitro in a sample from the patient. Preferably, the sample to be assessed / analyzed herein is a tumor tissue sample. A patient (or a patient group) is determined as being in need of a PD-L1 inhibitor cotherapy if a low or absent ER expression level and an expression level of programmed death ligand 1 (PD-L1) that is increased in comparison to a control and, optionally, an expression level of interferon-gamma (IFNγ) that is decreased in comparison to the control, is measured in vitro in said sample.

[0047] The term “ER” is an abbreviation of “Estrogen receptor”. Likewise, the terms “PD-L1” and “IFN-γ” are abbreviations of the terms “programmed death ligand” and “interferon-gamma”, respectively. Accordingly, the term “ER” can be used interchangeably herein with “Estrogen receptor”. Likewise, the terms “PD-L1” and “IFN-γ” can be used interchangeably herein with the terms “programmed death ligand” and “interferon-gamma”, respectively.

[0048] Preferably, the (tumor / biological) sample of the patient and / or the cancer to be treated is characterized by or associated with a low or absent estrogen receptor (ER) expression level. Preferably, the sample of the patient is a tumor sample. The ER expression level can be ER negative (ER(−)). The term “ER(−)” can be used herein interchangeably with the term “ER negative”.

[0049] “ER negative” expression level can be determined by routine and standard procedures as described, for example, in the Guideline on Hormone Receptor Testing in Breast Cancer S. Nofech-Mozes, E. Vella, S. Dhesy-Thind, and W. Hanna (A Quality Initiative of the Program in Evidence-Based Care (PEBC), Cancer Care Ontario (CCO); Report Date: Apr. 8, 2011). The Guidelines (and references cited therein) are incorporated by reference in its entirety herein. These Guidelines are available at world wide web atcancercare.on.ca) andPEBC Pathology & Laboratory Medicine page at:cancercare.on.ca / toolbox / qualityguidelines / clin-program / pathlabebs /

[0050] Routine and standard procedures for determining the “ER negative” expression level are described in these Guideline and also in the following references:

[0051] Nofech-Mozes S, Vella E T, Dhesy-Thind S, Hagerty K L, Mangu P B, Temin S, et al. Systematic review on hormone receptor testing in breast cancer. Applied Immunohistochem Mol Morphol. 2012 May; 20(3):214-63. doi: 10.1097 / PAI.0b013e318234aa12. Epub 2011 Nov. 11.

[0052] Nofech-Mozes S, Vella E T, Dhesy-Thind S, Hanna W M. Cancer Care Ontario guideline recommendations for hormone receptor testing in breast cancer. Clin Oncol (R Coll Radiol). Epub 2012 May 17.

[0053] “ER negative” expression may be determined by IHC (immunohistochemistry), if, for example the expression level of ER is low or absent and / or if the progesterone receptor (PR) expression level is low or absent. The abbreviation “PR” is used herein interchangeably with the term “progesterone receptor”. A sample or patients may be assessed as “ER negative” herein according to the following staining pattern (by IHC):

[0054] Only nuclear (not cytoplasmic) staining should be scored.

[0055] There are three categories for staining:

[0056] Positive: >10% staining for ER or PR

[0057] Low positive: 1% to 9% staining for ER or PR

[0058] Negative: <1% staining for ER and PR

[0059] Accordingly, a sample or patients may particularly be assessed as “ER negative” herein if the sample shows the following staining pattern by IHC: <1% staining for ER and PR.

[0060] Samples or patients may be assessed as “ER positive” herein if the sample shows a “positive” staining by IHC: ≥1% staining for ER or PR (i.e. more than 1% of the cells examined / assessed have estrogen receptors or progesterone receptors / show staining for estrogen receptors by IHC (immunohistochemistry).

[0061] Preferably, a sample or patient is assessed as “ER negative” herein if the sample shows the following staining pattern by IHC::<1% staining for ER (i.e. less than 1% of the cells examined / assessed have estrogen receptors / show staining for estrogen receptor(s) by IHC (immunohistochemistry). Most preferably, a sample or patients is / are assessed as “ER negative” if the nuclei in a tumor tissue sample show <1% staining for ER staining by IHC. Accordingly, from the three categories provided herein above, the assessment of “ER negative” is based on <1% staining for ER by IHC.

[0062] Likewise, “ER negative” expression can be determined by further methods routinely employed in the art. For example, “ER negative” may be determined if the mRNA / RNA expression level is low or absent. Routine methods to be used comprise, but are not limited to: Allred score, IRS, Remmele score or any other suitable biochemical detection method. A person skilled in the art is aware that the cut-off for such methods has to match the cut-off as defined above via IHC.

[0063] Nucleic acid sequences and amino acid sequences of Progesterone receptor (PR), Estrogen receptor (ER), of programmed death ligand 1 (PD-L1), and / or of interferon-gamma (IFNγ) to be used herein are well known and can be retrieved from databases like NCBI. Examplary sequences are provided herein (see for example SEQ ID NO: 38-51).

[0064] The methods and sample types used for establishing a cut-off value of a marker (like programmed death ligand 1 (PD-L1) and / or interferon-gamma (IFN-γ)) and for measuring the sample obtained from an individual or patient to be analyzed match each other or are the same. Cut-off values, i.e. values above which overexpression (e.g. increased expression of programmed death ligand 1 (PD-L1) in comparison to a control) is acknowledged can be obtained in a control group. Cut-off values, i.e. values below which decreased expression (e.g. decreased expression of interferon-gamma (IFN-7) in comparison to a control) is acknowledged can be obtained in a control group.

[0065] The control group on which the cut-off value is based is chosen to match the group of individuals / patients under investigation. In other words, if the method of the present invention is used to determine the need for PD-L1 cotherapy in patients with breast cancer or gastric cancer, respectively, the control group is also patients with breast cancer or gastric cancer, respectively. The control group used to establish the cut-off values for both, PDL-1 and IFN-γ, respectively), comprises at least 40, or at least 50, or at least 100 individuals / patients. An expression level or corresponding value above the cut-off is considered to represent overexpression and a value at or below the cut-off is considered as decreased expression.

[0066] In one embodiment, the “IFN-γ” expression level in a tumor tissue sample from an individual / patient is compared to a cut-off value. A value above the cut-off is considered to represent overexpression of IFN-γ and a value at or below the cut-off is considered as decreased expression of IFN-γ. In one embodiment the decreased expression is acknowledged if the expression level for IFN-γ is at or below the value of the highest quintile, quartile or tertile, respectively, as established in the control group. In one embodiment the cut-off for IFN-γ is the highest tertile. In one embodiment the cut-off value is a value between the 70th and the 80th percentile. In one embodiment the cut-off value for IFN-γ is the 73rd percentile, i.e a value above this cut-off is considered to represent overexpression of IFN-γ and a value at or below the 73rd percentile is considered as decreased expression of IFN-γ. In one embodiment, individuals / patients are determined as being in need of a PD-L1 cotherapy, if IFN-γ expression in a sample (like a tumor tissue sample) is decreased (i.e. below or at the IFN-γ cut-off value) In one embodiment individuals / patients are determined as not being in need of a PDL-1 cotherapy, if IFN-γ is overexpressed (i.e. above the IFN-γ cut-off value as described above).

[0067] In one embodiment the PD-L1 expression level, in a tumor tissue sample from an individual / patient is compared to a cut-off value. A value above the cut-off is considered to represent overexpression of PD-L1 and a value at or below the cut-off is considered as decreased expression of PD-L1. In one embodiment overexpression for PDL-1 is acknowledged if the expression level for PDL-1 is above a cut-off value between the 50th percentile and the 75th percentile, as established in a control group. In one embodiment overexpression for PDL-1 is acknowledged if the expression level for PDL-1 is above a cut-off value between the 50th percentile and the 70th percentile, of the control group. In one embodiment individuals / patients are determined as being in need of a PDL-1 cotherapy, if PDL-1 is overexpressed (i.e. the PDL-1 expression level determined is above the PDL-1 cut-off value).

[0068] In one further embodiment overexpression for PDL-1 is established in the sub-group of individuals / patients having a decreased expression level of IFN-γ in a tumor tissue sample. In one embodiment overexpression for PDL-1 is acknowledged if the expression level for PDL-1 is above a cut-off value between the 40th percentile and the 65th percentile, as established in this sub-group. In one embodiment overexpression for PDL-1 is acknowledged if the expression level for PDL-1 is above a cut-off value between the 50th percentile and the 60th percentile, as established in this sub-group. In one embodiment individuals / patients are determined as being in need of a PDL-1 cotherapy, if the PDL-1 expression level in the sub-group with decreased expression of IFN-γ is above the 54th percentile.

[0069] In one embodiment, individuals / patients are determined as being in need of a PDL-1 cotherapy, if IFN-γ expression in a tumor tissue sample is decreased (i.e. below or at the IFN-γ cut-off value) and PDL-1 is overexpressed (i.e. above the PDL-1 cut-off value).

[0070] The term “expression level of programmed death ligand 1 (PD-L1) that is increased in comparison to a control” can be used interchangeably herein with “expression level of programmed death ligand 1 (PD-L1) above the PDL-1 cut-off value” as defined and explained herein above.

[0071] The term “expression level of interferon-gamma (IFNγ) that is decreased in comparison to a control” can be used interchangeably herein with “expression level of interferon-gamma (IFNγ) below or at the IFNγ cut-off value”.

[0072] The present invention relates to the following aspects.

[0073] The present invention relates to a method of determining the need of a cancer patient for a PD-L1 inhibitor cotherapy, (i) wherein therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated for the patient or (ii) wherein the patient is undergoing therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent, the method comprising the steps of

[0074] a) measuring in vitro in a sample from said patient the expression level of Estrogen receptor (ER), of programmed death ligand 1 (PD-L1), and of interferon-gamma (IFNγ);

[0075] b) determining a patient as being in need of a PD-L1 inhibitor cotherapy if a low or absent ER expression level (like ER(−) / ER-negative), an expression level of programmed death ligand 1 (PD-L1) above the PDL-1 cut-off value and an expression level of interferon-gamma (IFNγ) below or at the IFNγ cut-off value is measured in step (a).

[0076] The present invention relates to a method of treating a cancer in a cancer patient for whom therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated, the method comprising selecting a cancer patient whose cancer is determined to have a low or absent ER expression level (like ER(−) / ER-negative) and to have an expression level of programmed death ligand 1 (PD-L1) above the PDL-1 cut-off value and to have an expression level of interferon-gamma (IFNγ) below or at the IFNγ cut-off value, and administering to the patient an effective amount of a modulator of the HER2 / neu (ErbB2) signaling pathway, of a chemotherapeutic agent and of a programmed death ligand 1 (PD-L1) inhibitor.

[0077] The present invention relates to a method of treating a cancer in a cancer patient who is undergoing therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent, the method comprising selecting a cancer patient whose cancer is determined to have a low or absent ER expression level (like ER(−) / ER-negative) and to have an expression level of programmed death ligand 1 (PD-L1) above the PDL-1 cut-off value and to have an expression level of interferon-gamma (IFNγ) below or at the IFNγ cut-off value, and administering to the patient an effective amount of a programmed death ligand 1 (PD-L1) inhibitor.

[0078] The present invention relates to a pharmaceutical composition comprising a modulator of the HER2 / neu (ErbB2) signaling pathway, and an inhibitor of programmed death ligand 1 (PD-L1) for use in the treatment of cancer, whereby said cancer is determined to have a low or absent ER expression level (like ER(−) / ER-negative) and to have an expression level of programmed death ligand 1 (PD-L1) above the PDL-1 cut-off value and to have an expression level of interferon-gamma (IFNγ) below or at the IFNγ cut-off value.

[0079] All explanations and definitions given herein for “PD-L1 inhibitor”, “PD-L1 inhibitor cotherapy”, “cancer”, “cancer patient”, “modulator of the HER2 / neu (ErbB2) signaling pathway”, “chemotherapeutic agent”, “sample”, “expression level” and the like apply, mutatis mutandis, to the above aspects of the present invention.

[0080] The expression level of Estrogen receptor (ER), of programmed death ligand 1 (PD-L1), and of interferon-gamma (IFNγ) in a sample from the patient may be measured in vitro simultaneously or subsequently in any combination. For example, the expression level of Estrogen receptor (ER), of programmed death ligand 1 (PD-L1), and of interferon-gamma (IFNγ) may be measured simultaneously. The expression level of Estrogen receptor (ER) may be measured first, followed by the measurement of programmed death ligand 1 (PD-L1) and of interferon-gamma (IFNγ). The expression level of programmed death ligand 1 (PD-L1) may be measured first, followed by the (simultaneous or subsequent) measurement of Estrogen receptor (ER) and of interferon-gamma (IFNγ). The expression level of interferon-gamma (IFNγ) may be measured first, followed by the (simultaneous or subsequent) measurement of Estrogen receptor (ER) and of programmed death ligand 1 (PD-L1). Any order / combination of the measurement of the expression level of Estrogen receptor (ER), of programmed death ligand 1 (PD-L1), and of interferon-gamma (IFNγ) in a sample from the patient is envisaged and comprised herein.

[0081] Herein contemplated is a determination of a patient as being in need of a PD-L1 inhibitor cotherapy if, in a first step (1) a low or absent ER expression level (like ER(−) / ER-negative) is measured, and if, in a second step (2) an expression level of interferon-gamma (IFNγ) below or at the IFNγ cut-off value is measured and if, in a third step (3) an expression level of programmed death ligand 1 (PD-L1) above the PDL-1 cut-off value is measured.

[0082] The present invention relates to the following aspects:

[0083] The present invention relates to a method of determining the need of a cancer patient for a PD-L1 inhibitor cotherapy, (i) wherein therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated for the patient or (ii) wherein the patient is undergoing therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent, the method comprising the steps of

[0084] a) measuring in vitro in a sample from said patient the expression level of Estrogen receptor (ER), of programmed death ligand 1 (PD-L1), and of interferon-gamma (IFNγ);

[0085] b) determining a patient as being in need of a PD-L1 inhibitor cotherapy if, in a first step (1) a low or absent ER expression level (like ER(−) / ER-negative) is measured, and if in a second step (2) an expression level of interferon-gamma (IFNγ) below or at the IFNγ cut-off value is measured and if in a third step (3) an expression level of programmed death ligand 1 (PD-L1) above the PDL-1 cut-off value is measured.

[0086] The present invention relates to a method of treating a cancer in a cancer patient for whom therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated, the method comprising selecting a cancer patient whose cancer is determined to have in a first step (1) a low or absent ER expression level (like ER(−) / ER-negative) and in a second step (2) to have an expression level of interferon-gamma (IFNγ) below or at the IFNγ cut-off value, and in a third step (3) to have an expression level of programmed death ligand 1 (PD-L1) above the PDL-1 cut-off value, and administering to the patient an effective amount of a modulator of the HER2 / neu (ErbB2) signaling pathway, of a chemotherapeutic agent and of a programmed death ligand 1 (PD-L1) inhibitor.

[0087] The present invention relates to a method of treating a cancer in a cancer patient who is undergoing therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent, the method comprising selecting a cancer patient whose cancer is determined to have in a first step (1) a low or absent ER expression level (like ER(−) / ER-negative) and in a second step (2) to have an expression level of interferon-gamma (IFNγ) below or at the IFNγ cut-off value, and in a third step (3) to have an expression level of programmed death ligand 1 (PD-L1) above the PDL-1 cut-off value, and administering to the patient an effective amount of a programmed death ligand 1 (PD-L1) inhibitor.

[0088] The present invention relates to a pharmaceutical composition comprising a modulator of the HER2 / neu (ErbB2) signaling pathway, and an inhibitor of programmed death ligand 1 (PD-L1) for use in the treatment of cancer, whereby said cancer is determined to have a low or absent ER expression level (like ER(−) / ER-negative), to have an expression level of interferon-gamma (IFNγ) below or at the IFNγ cut-off value, to have an expression level of programmed death ligand 1 (PD-L1) above the PDL-1 cut-off value.

[0089] All explanations and definitions given herein for “PD-L1 inhibitor”, “PD-L1 inhibitor cotherapy”, “cancer”, “cancer patient”, “modulator of the HER2 / neu (ErbB2) signaling pathway”, “chemotherapeutic agent”, “sample”, “expression level” and the like apply, mutatis mutandis, to the above aspects of the present invention.

[0090] The following relates to an exemplary cut-off value allowing determining a patient as being in need of a PD-L1 inhibitor cotherapy in accordance with the present invention. It can be easily determined by routine techniques (such as Affymetrix) whether the expression level of PD-L1 and / or IFN-gamma in a sample from a patient is below or above such cut-off values.

[0091] If a gene expression analysis gives a result for IFN-gamma expression higher or equal to 4.8 no combination treatment (HER2-targeted and PDL1-targeted) is recommended and no further PDL1 assessment is necessary. If a gene expression analysis gives a result for IFN-gamma lower than 4.8 a parallel assessment of PDL-1 is necessary. If PDL-1 gene expression analysis then gives a result of higher or equal to 5.3 a combination treatment (HER2-targeted and PDL1-targeted) is recommended. This exemplary protocol is illustrated in FIG. 19.

[0092] In this context Affymetrix can be performed as follows: Total RNA from tumor cells was extracted FFPE tumor sections using Light Cycler Pertuzumab FFPET RNA Kit (Roche Diagnostics). RNA was processed for hybridization using the WT-Ovation FFPE System V2 (Nugen) and hybridized to Affymetrix GeneChip® Human Genome U133 Plus 2.0 Arrays. Hybridized arrays were washed and stained on Affymetrix Fluidics Station 450 and scanned with an Affymetrix GeneChip® Scanner 3000 7G.

[0093] As mentioned the expression level of PD-L1 and / or IFN-gamma in a sample from a patient can be determined by routine techniques, such as Affymetrix. The following relates an exemplary protocol for such a determination (also termed herein Gene Expression Profiling):

[0094] The tumor biopsy samples can be profiled for gene expression on AFFYMETRIX HG-U133Plus 2 whole Human Genome microarray platform. Roche HighPure RNA extraction, NuGen amplification and standard AFFYMETRIX hybridization and scanning protocols can be used. These protocols etc. are incorporated herein by reference. All array scans usually pass standard AFFYMETRIX QC.

[0095] Robust Multiarray algorithm (RMA) can be used for preprocessing of raw signals (Irizarry et al, 2003. World wide web at ncbi.nlm.nih.gov / pubmed / 12925520; incorporated herein by reference). All probe sets available for the genes of interest can be retrieved as reported below. For gene CD274, when several probe sets were available to represent this gene, the probe set with the highest average expression value (defined as an arithmetical average of expression of a given probe set) was selected to represent the gene:

[0096] CD274 (PDL1)

[0097] 223834_at selected for PDL1

[0098] 227458_at

[0099] The selected probe set corresponds to the last exon / 3′UTR of the gene and captures all known RefSEq mRNAs (see FIG. 6)

[0100] IFNG

[0101] 210354_at

[0102] This probe set also represents the last exon / 3′UTR of the gene and captures all known RefSEq mRNAs (see FIG. 7)

[0103] In accordance with the above, the expression level of Interferon-gamma may be measured prior to the expression level of Estrogen receptor (ER) and / or prior to the expression level of programmed death ligand 1 (PD-L1). The step of measuring the expression level of Estrogen receptor (ER) and of programmed death ligand 1 (PD-L1) may even be absent.

[0104] As shown in the appended Example, PD-L1 cotherapy can, for example, not be recommended if the expression level of interferon-gamma (IFNγ) is higher or equal to (about) 4.8 as determined by routine methods like Affymetrix.

[0105] Accordingly, the present invention provides a method of determining the need of a cancer patient for a PD-L1 inhibitor cotherapy, wherein therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated for the patient or wherein the patient is undergoing therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent, the method comprising the steps

[0106] (a) measuring in vitro in a sample from said patient the expression level of interferon-gamma (IFNγ)

[0107] (b) determining a patient as being not in need of a PD-L1 inhibitor cotherapy if the expression level of interferon-gamma (IFNγ) is higher or equal to (about) 4.8 as determined by routine methods like Affymetrix in step (a).

[0108] If the expression level of interferon-gamma (IFNγ) is lower than (about) 4.8 as determined by routine methods like Affymetrix, the expression level of programmed death ligand 1 (PD-L1) and, optionally, Estrogen receptor (ER) can be measured in vitro in a sample from said patient.

[0109] Accordingly, the present invention provides a method of determining the need of a cancer patient for a PD-L1 inhibitor cotherapy, wherein therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated for the patient or wherein the patient is undergoing therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent, the method comprising the steps

[0110] (a) measuring in vitro in a sample from said patient the expression level of interferon-gamma (IFNγ), Estrogen receptor (ER) and of programmed death ligand 1 (PD-L1),

[0111] (b) determining a patient as being in need of a PD-L1 inhibitor cotherapy if the expression level of interferon-gamma (IFNγ) is lower than (about) 4.8 as determined by routine methods like Affymetrix, and if a low or absent ER expression level and, optionally, an expression level of programmed death ligand 1 (PD-L1) that is increased in comparison to a control is measured in step (a).

[0112] A patient can be determined in accordance with the present invention to be in need of PD-L1 inhibitor cotherapy if the expression level of programmed death ligand 1 (PD-L1) measured in the sample from the patient is increased in comparison to a control. For example, the expression level of programmed death ligand 1 (PD-L1) can be higher or equal to (about) 5.3 determined by routine methods like Affymetrix.

[0113] All explanations and definitions given herein for “PD-L1 inhibitor”, “PD-L1 inhibitor cotherapy”, “cancer”, “cancer patient”, “modulator of the HER2 / neu (ErbB2) signaling pathway”, “chemotherapeutic agent”, “sample”, “expression level” and the like as given herein apply, mutatis mutandis, in this context.

[0114] Accordingly, the present invention relates to a method of treating a cancer in a cancer patient for whom therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated, the method comprising selecting a cancer patient whose cancer is determined to have a low or absent ER expression level and to have an increased expression level of programmed death ligand 1 (PD-L1) in comparison to a control, and an expression level of interferon-gamma (IFNγ) that is lower than (about) 4.8 as determined by routine methods like Affymetrix, and administering to the patient an effective amount of a modulator of the HER2 / neu (ErbB2) signaling pathway, of a chemotherapeutic agent and of a programmed death ligand 1 (PD-L1) inhibitor.

[0115] Furthermore, the present invention relates to a method of treating a cancer in a cancer patient who is undergoing therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent, the method comprising selecting a cancer patient whose cancer is determined to have a low or absent ER expression level and to have an increased expression level of programmed death ligand 1 (PD-L1) in comparison to a control, and to have an expression level of interferon-gamma (IFNγ) that is lower than (about) 4.8 as determined by routine methods like Affymetrix, and administering to the patient an effective amount of a programmed death ligand 1 (PD-L1) inhibitor.

[0116] A pharmaceutical composition is provided comprising a modulator of the HER2 / neu (ErbB2) signaling pathway, and an inhibitor of programmed death ligand 1 (PD-L1) for use in the treatment of cancer, whereby said cancer is determined to have a low or absent ER expression level and to have an increased expression level of programmed death ligand 1 (PD-L1) in comparison to a control, and an expression level of interferon-gamma (IFNγ) that is lower than (about) 4.8 as determined by routine methods like Affymetrix.

[0117] The pharmaceutical composition for use in the treatment of cancer may further comprise a chemotherapeutic agent.

[0118] In accordance with the above, the herein provided methods may comprise a step of measuring the expression level of Interferon-gamma (IFNγ) in said sample and determining a patient as being in need of a PD-L1 inhibitor cotherapy if an expression level of interferon-gamma (IFNγ) that is decreased in comparison to the control is measured. For example, a “decreased expression level” of interferon-gamma (IFNγ) may be an expression level lower than (about) 4.8 as determined by routine methods like Affymetrix. Accordingly, the cancer that is determined to have a decreased expression level of interferon-gamma (IFNγ) in comparison to the control may be determined to have an expression level of interferon-gamma (IFNγ) that is lower than (about) 4.8 as determined by routine methods like Affymetrix,

[0119] It is envisaged herein that the expression level may be reflected in the activity of the gene product / protein. Accordingly, also the activity of ER, PD-L1 and / or IFN-γ can be measured and evaluated in addition or in the alternative to the expression level in accordance with the present invention. A person skilled in the art is aware of corresponding means and methods for detecting and evaluating the ER, PD-L1 and IFN-γ expression level and / or activity. Exemplary methods to be used include but are not limited to molecular assessments such as Western Blots, Northern Blots, Real-Time PCR and the like. Such methods are described herein in detail.

[0120] The expression level of ER, PD-L1 and / or IFN-γ may be the mRNA expression level of ER, PD-L1 and / or IFN-γ. If the gene product is an RNA, in particular an mRNA (e.g. unspliced, partially spliced or spliced mRNA), determination can be performed by taking advantage of northern blotting techniques, in situ hybridization, hybridization on microarrays or DNA chips equipped with one or more probes or probe sets specific for mRNA transcripts or PCR techniques, like, quantitative PCR techniques, such as Real time PCR. These and other suitable methods for binding (specific) mRNA are well known in the art and are, for example, described in Sambrook and Russell (2001, loc. cit.). A skilled person is capable of determining the amount of the component, in particular said gene products, by taking advantage of a correlation, preferably a linear correlation, between the intensity of a detection signal and the amount of the gene product to be determined.

[0121] The expression level may be the protein expression level of ER, PD-L1 and / or IFN-γ. Quantification of the protein expression level can be performed by taking advantage of the well known techniques such as western blotting techniques, immunoassays, gel- or blot-based methods, IHC, mass spectrometry, flow cytometry, FACS and the like. Generally, a person skilled in the art is aware of methods for the quantitation of (a) polypeptide(s) / protein(s). Amounts of purified polypeptide in solution can be determined by physical methods, e.g. photometry. Methods of quantifying a particular polypeptide in a mixture may rely on specific binding, e.g of antibodies. Specific detection and quantitation methods exploiting the specificity of antibodies comprise for example immunohistochemistry (in situ). Western blotting combines separation of a mixture of proteins by electrophoresis and specific detection with antibodies. Electrophoresis may be multi-dimensional such as 2D electrophoresis. Usually, polypeptides are separated in 2D electrophoresis by their apparent molecular weight along one dimension and by their isoelectric point along the other direction. Alternatively, protein quantitation methods may involve but are not limited to mass spectrometry or enzyme-linked immunosorbant assay methods.

[0122] Also, the use of high throughput screening (HTS) is envisaged in the context of the present invention. Suitable (HTS) approaches are known in the art. A person skilled in the art is readily in the position to adapt such protocols or known HTS approaches to the performance of the methods of the present invention. Such assays are usually performed in liquid phase, wherein for each cell / tissue / cell culture to be tested at least one reaction batch is made. Typical containers to be used are micro titer plates having for example, 384, 1536, or 3456 wells (i.e. multiples of the “original” 96 reaction vessels). Robotics, data processing and control software, and sensitive detectors, are further commonly used components of a HTS device. Often robot systems are used to transport micro titer plates from station to station for addition and mixing of sample(s) and reagent(s), incubating the reagents and final readout (detection). Usually, HTS can be used in the simultaneous preparation, incubation and analysis of many plates. The assay can be performed in a single reaction (which is usually preferred), may, however, also comprise washing and / or transfer steps. Detection can be performed taking advantage of radioactivity, luminescence or fluorescence, like fluorescence-resonance-energy transfer (FRET) and fluorescence polarisation (FP) and the like. The biological samples described herein can also be used in such a context. In particular, cellular assays and in vivo assays can be employed in HTS. Cellular assays may also comprise cellular extracts, i.e. extracts from cells, tissues and the like. However, preferred herein is the use of cell(s) or tissue(s) as biological sample (in particular a sample obtained from a patient / subject suffering or being prone to suffer from cancer), whereas in vivo assays are particularly useful in the validation of modulators / inhibitors / chemotherapeutic agents to be used herein. Depending on the results of a first assay, follow up assays can be performed by re-running the experiment to collect further data on a narrowed set (e.g. samples found “positive” in the first assay), confirming and refining observations.

[0123] As used in context of the methods of the present invention, a non-limiting example of a “control” is preferably a control from a patient who is not in need of a PD-L1 inhibitor cotherapy, for example a sample / cell / tissue obtained from one or more healthy subjects or one or more patients that suffer from a cancer / tumor and are known to be not in need of a PD-L1 inhibitor cotherapy treatment. For example, such a control (sample) may be from a patient who does not benefit from additional PD-L1 inhibitor cotherapy. Another non-limiting example of a “control” is an “internal standard”, for example a mixture of purified or synthetically produced proteins and / or peptides or RNA, where the amounts of each protein / peptide / RNA is gauged by using the control described above.

[0124] A further non-limiting example of a “control” may be a “healthy” control, for example a sample / cell / tissue obtained from a healthy subject or patient that is not suffering from a cancer / tumor or a cell obtained from such a subject. In accordance with the above, the reference or control expression level of ER, PD-L1 and / or IFN-γ is that determined in (a sample of) the corresponding healthy control subject / patient, i.e. it is the “normal” status of ER, PD-L1 and / or IFN-γ. The control may also be a sample / cell / tissue obtained from the individual or patient suspected of suffering from the cancer provided that the sample / cell / tissue does not contain tumor or cancer cells. In a further alternative, the “control” may be a sample / cell / tissue obtained from an individual or patient suffering from the cancer, that has been obtained prior to the development or diagnosis of said cancer.

[0125] The sample to be assessed in accordance with the herein provided methods may comprise non-diseased cells and / or diseased cells, i.e. non-cancerous cells and / or cancerous cells. However, the content of cancerous cells among non-cancerous cells should be higher than for example 50%. The sample may also (or even solely) comprise cancer / tumor cell(s), such as breast cancer / tumor cell(s). The term “sample” shall generally mean any biological sample obtained from a patient's tumor. The sample may be a tissue resection or a tissue biopsy. The sample may also be a metastatic lesion or a section of a metastatic lesion or a blood sample known or suspected to comprise circulating tumor cells. In accordance with the above, the biological sample may comprise cancer cells and to a certain extent i.e. less than for example 50% non-cancer cells (other cells). The skilled pathologist is able to differentiate cancer cells from normal tissue cells. Methods for obtaining tissue biopsies, tissue resections and body fluids and the like from mammals, such as humans, are well known in the art.

[0126] As explained above, the cancer patient who is determined to be in need of PD-L1 inhibitor cotherapy in accordance with the present invention is undergoing therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent or such a therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated for the patient. Therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent is indicated for patients with “HER2-positive cancer”, like a patient that is suspected to suffer from a HER2-positive cancer, suffering from a HER2-positive cancer or being prone to suffer from a HER2-positive cancer. Preferably, the cancer to be treated is in accordance with the present invention a “HER2-positive cancer”, particularly a “HER2-positive breast cancer”. A “HER2-positive cancer” can be a “HER2-positive breast cancer” or a “HER2-positive gastric cancer”. Further, the HER2-positive cancer may be ovarian cancer, lung cancer, colorectal cancer, kidney cancer, bone cancer, bone marrow cancer, bladder cancer, skin cancer, prostate cancer, esophagus cancer, salivary gland cancer, pancreas cancer, liver cancer, head and neck cancer, CNS (especially brain) cancer, cervix cancer, cartilage cancer, colon cancer, genitourinary cancer, gastrointestinal tract cancer, pancreas cancer, synovium cancer, testis cancer, thymus cancer, thyroid cancer and uterine cancer.

[0127] The term “HER2-positive cancer” as used herein refers to a cancer / tumorous tissue etc. which comprises cancer cells which have higher than normal levels of HER2. For the purpose of the present invention, “HER2-positive cancer” has an immunohistochemistry (IHC) score of at least 2+ and / or an in situ hybridization (ISH) amplification ratio ≥2.0 (i.e. is ISH-positive). Accordingly, HER2-positive cancer is present if a high HER2 (protein) expression level detected e.g. by immunohistochemical methods and / or HER2 gene amplification detected by in-situ-hybridization (ISH positive, like a HER2 gene copy number higher than 4 copies of the HER2 gene per tumor cell or ratio of ≥2.0 for the number of HER2 gene copies to the number of signals for CEP17.) is found in samples obtained from the patients such as breast tissue biopsies or breast tissue resections or in tissue derived from metastatic sites. In one embodiment “HER2-positive cancer” has an immunohistochemistry (IHC) score of HER2(3+) and / or is ISH positive.

[0128] The expression level of HER2 may be detected by an immunohistochemical method, whereas said HER2 gene amplification status can be measured with in situ hybridization methods, like fluorescence in situ hybridization techniques (FISH). Corresponding assays and kits are well known in the art, for protein expression assays as well as for the detection of gene amplifications. Alternatively, other methods like qRT-PCR might be used to detect levels of HER2 gene expression.

[0129] The expression level of HER2 can, inter alia, be detected by an immunohistochemical method. Such methods are well known in the art and corresponding commercial kits are available. Exemplary kits which may be used in accordance with the present invention are, inter alia, HerceptTest™ produced and distributed by the company Dako or the test called Ventana Pathway™. The level of HER2 protein expression may be assessed by using the reagents provided with and following the protocol of the HercepTest™. A skilled person will be aware of further means and methods for determining the expression level of HER2 by immunohistochemical methods; see for example WO 2005 / 117553. Therefore, the expression level of HER2 can be easily and reproducibly determined by a person skilled in the art without undue burden. However, to ensure accurate and reproducible results, the testing must be performed in a specialized laboratory, which can ensure validation of the testing procedures. The expression level of HER2 can be classified in a low expression level, an intermediate expression level and a high expression level. It is preferred in context of this invention that HER2-positive disease is defined by a strong expression level of HER2 (e.g. HER2(3+) by IHC), for example determined in a sample of a cancer patient.

[0130] The recommended scoring system to evaluate the IHC staining patterns which reflects the expression levels of HER2 designated herein HER2(0), HER2(+), HER2(++) and HER2(+++), is as follows:StainingHER2IntensityoverexpressionScoreStaining Patternassessment0 No staining is observed or negativemembrane staining is observedin <10% of the tumor cells1+A faint / barely perceptible negativemembrane staining is detectedin >10% of the tumor cells, the cells are only stained inpart of their membrane.2+A weak to moderate complete weak to staining is detected in >10%moderateof the tumor cells.overexpression.3+A strong complete membrane strongstaining is detected in >10%overexpression.of the tumor cells.

[0131] The above IHC staining patterns are routinely used in determining HER2-positive breast cancer. The terms HER2(+), HER2(++) and HER2(+++) used herein are equivalent to the terms HER2(1+), HER2(2+) and HER2(3+). A “low protein expression level” used in context of this invention corresponds to a 0 or 1+ score (“negative assessment” according to the table shown herein above), an “weak to moderate protein expression level” corresponds to a 2+ score (“weak to moderate overexpression”, see the table above) and a “high protein expression level” corresponds to a 3+ score (“strong overexpression”, see the table above). As described herein above in detail, the evaluation of the protein expression level (i.e. the scoring system as shown in the table) is based on results obtained by immunohistochemical methods. As a standard or routinely, the HER-2 status is, accordingly, performed by immunohistochemistry with one of two FDA-approved commercial kits available; namely the Dako Herceptest™ and the Ventana Pathway™. These are semi-quantitative assays which stratify expression levels into 0 (<20,000 receptors per cell, no expression visible by IHC staining), 1+(˜100,000 receptors per cell, partial membrane staining, <10% of cells overexpressing HER-2), 2+(˜500,000 receptors per cell, light to moderate complete membrane staining, >10% of cells overexpressing HER-2), and 3+(˜2,000,000 receptors per cell, strong complete membrane staining, >10% of cells overexpressing HER-2).

[0132] Alternatively, further methods for the evaluation of the protein expression level of HER2 may be used, e.g. Western Blots, ELISA-based detection systems and so on.

[0133] A HER2-positive cancer may also be diagnosed by assessing the gene amplification status of HER2. HER2-positive cancer is, accordingly, diagnosed if this assessment by ISH is positive. In accordance with this assessment, a HER2-positive cancer may, inter alia, relate to an average HER2 gene copy number higher than 4 copies of the HER2 gene per tumor cell (for those test systems without an internal centromere control probe) or to a HER2 / CEP17 ratio of >=2.0 (for those test systems using an internal chromosome 17 centromere control probe). In other words, the HER2-positive cancer may, inter alia, relate to a HER2 gene copy number greater than 4. The amplification level of the HER2 gene may easily be identified by in situ hybridization (ISH) like fluorescent in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and silver in situ hybridization (SISH). These methods are known to the skilled artisan. The principles of these methods can be deduced from standard text books. Commercial kits for the determination of the HER2 gene amplification status by in situ hybridization are available.

[0134] The below IHC staining patterns are recommended for determining HER2-positive gastric cancer (see Dako Herceptest package insert).of Hercep Test™ stained biopsies a cluster of at least 5 stained tumor cells is recommended. A cluster of at least 5 stained tumor cells consists of 5 connected HER2 stained tumor cells.TABLE 9Interpretation and scoring of HER2 immunohistochemical stainingHER2Over-Surgical Specimen-Biopsy Specimen-expressionScoreStaining PatternStaining PatternAssessment0 No reactivity No reactivity or Negativeor membranousno membranousreactivity in reactivity in any <10% of tumor cells(or <5 clustered)tumor cell1+Faint / barely perceptibleTumor cell cluster Negativemembranous (≥5 cells) with areactivity in ≥10% offaint / barely perceptible tumor cells, cells membranousare reactive only inreactivity irrespective part of their membraneof percentageof tumor cells stained2+Weak to moderate Tumor cell cluster Equivocalcomplete, basolateral (≥5 cells) with a weak or lateral membranousto moderate complete,reactivity in ≥10% basolateral or of tumor cellslateral membranousreactivity irrespective of percentageof tumor cells stained3+Strong complete, Tumor cell cluster Positivebasolateral or(≥5 cells) with a strong lateral membranous complete, basolateral orreactivitylateral membranous in ≥10% of tumor cellsreactivity irrespective of percentage of tumorcells stainedGuidelines based on Hofmann et al. (40).More refined IHC staining patterns for determining HER2-positive gastric cancer is as follows:HER2StainingOver-IntensitySurgical specimen-Biopsy specimen-expressionScorestaining patternstaining patternAssessment0 No reactivity or noNo reactivity or noNegativemembranous reactivitymembranous in <10% of tumour cellsreactivity in anytumour cell1+Faint / barely perceptibleTumour cell cluster (≥5Negativemembranous reactivitycells) with a faint / barelyin ≥10% of tumour cells;perceptible membranouscells are reactive only inreactivity irrespective ofpart of their membranepercentage of tumour cells stained2+Weak to moderateTumour cell cluster (≥5Equivocalcomplete, basolateral orcells) with a weak tolateral membranousmoderate complete,reactivity in ≥10% ofbasolateral or lateraltumour cellsmembranous reactivityirrespective of percentage oftumour cells stained3+Strong complete, Tumour cell cluster (≥5Positivebasolateralcells) with a strongor lateral membranouscomplete, basolateral orreactivity in ≥10% oflateral membranous tumour cellsreactivity irrespective of percentage oftumour cells stainedAs indicated above, the HER2 positive cancer to be treated in accordance with the present invention may be breast cancer, such early stage breast cancer. The term “early-stage breast cancer” as used herein refers to breast cancer that has not spread beyond the breast or the axillary lymph nodes. Such cancer can be generally treated with neoadjuvant or adjuvant therapy. The term “neoadjuvant therapy” as used herein refers to systemic therapy given prior to surgery. The term “adjuvant therapy” refers to systemic therapy given after surgery. In accordance with the above, treatment may be neoadjuvant or adjuvant therapy of early-stage breast cancer.

[0137] In accordance with the above, the sample to be assessed can be (obtained) from a patient with HER2-positive cancer as defined above. For example, the sample may be obtained from a tumorous tissue, (a) tumor(s) and, accordingly, is (a) tumor cell(s) or (a) tumor tissue(s) suspected of being HER2-positive tumour, like a breast tumor and the like. A person skilled in the art is in the position to identify such tumors and / or individuals / patients suffering from corresponding cancer using standard techniques known in the art and methods disclosed herein. Generally, said tumor cell or cancer cell may be obtained from any biological source / organism, particularly any biological source / organism, suffering from the above-mentioned cancer. In context of this invention particular useful cells are, preferably, human cells. These cells can be obtained from e.g. biopsies or from biological samples. The tumor / cancer / tumor cell / cancer cell is a solid tumor / cancer / tumor cell / cancer cell. In accordance with the above, the cancer / tumor cell may be a breast cancer / tumor cell or said sample comprises a cancer / tumor cell, such as a breast cancer / tumor cell. In line with the above, said tumor / cancer may be a breast tumor / cancer.

[0138] The modulator of the HER2 / neu (ErbB2) signaling pathway may be an inhibitor of HER2, for example, a HER dimerization / signaling inhibitor. The HER dimerization inhibitor may be a HER2 dimerization inhibitor. The HER dimerization inhibitor may inhibit HER heterodimerization or HER homodimerization. The HER dimerization inhibitor may be an anti-HER antibody. The term “antibody” herein is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity. Also human and humanized as well as CDR-grafted antibodies are comprised within the term “antibody”.

[0139] The HER antibody may bind to a HER receptor selected from the group consisting of EGFR, HER2 and HER3. Preferably, the antibody binds to HER2. The anti HER2 antibody may bind to domain II of HER2 extracellular domain. The antibody may bind to a junction between domains I, II and III of HER2 extracellular domain. The anti HER2 antibody may be Pertuzumab.

[0140] For the purposes herein, “Pertuzumab” and “rhuMAb 2C4”, which are used interchangeably, refer to an antibody comprising the variable light and variable heavy domains (amino acid sequences thereof shown in SEQ ID Nos. 5 and 6, respectively, as depicted in FIG. 2). The variable light and variable heavy domains of variant 574 / Pertuzumab are also shown in FIG. 2 (amino acid sequences thereof shown in SEQ ID Nos. 7 and 8, respectively, as depicted in FIG. 2). Where Pertuzumab is an intact antibody, it preferably comprises an IgG1 antibody; in one embodiment comprising the light chain amino acid sequence in it preferably comprises the light chain and heavy chain amino acid sequences, respectively, as shown in FIGS. 3A / 3B and 5A / 5B (FIG. 5A / 5B show the light chain and heavy chain amino acid sequences of a variant Pertuzumab). The heavy chain amino acid sequences of Pertuzumab as shown in FIG. 3B may optionally comprise an additional amino acid “K” at position 449 at the C-terminus. The antibody is optionally produced by recombinant Chinese Hamster Ovary (CHO) cells. The terms “Pertuzumab” and “rhuMAb 2C4” herein cover biosimilar versions of the drug with the United States Adopted Name (USAN) or International Nonproprietary Name (INN): Pertuzumab. Again, corresponding sequences are shown in FIGS. 2 to 5.

[0141] The modulator of the HER2 / neu (ErbB2) signaling pathway may be an inhibitor of HER shedding, for example a HER2 shedding inhibitor. The inhibitor of HER shedding may inhibit HER heterodimerization or HER homodimerization. Said inhibitor of HER shedding may be an anti-HER antibody.

[0142] The anti-HER antibody may bind to a HER receptor selected from the group consisting of EGFR, HER2 and HER3. Preferably, the antibody binds to HER2. The HER2 antibody may bind to sub-domain IV of the HER2 extracellular domain. Preferably, the HER2 antibody is Herceptin™ / Trastuzumab.

[0143] For the purposes herein, “Herceptin™” / “Trastuzumab” and “rhuMAb4D5-8”, which are used interchangeably, refer to an antibody comprising the variable light domains and variable heavy domains (amino acid sequences thereof are shown in FIG. 4, respectively; the domain is indicated by arrows). Where Trastuzumab is an intact antibody, it preferably comprises an IgG1 antibody; in one embodiment comprising the light chain amino and the heavy chain amino acid sequence as shown in FIG. 4. The antibody is optionally produced by Chinese Hamster Ovary (CHO) cells. The terms “Trastuzumab” and “rhuMAb4D5-8” herein cover biosimilar versions of the drug with the United States Adopted Name (USAN) or International Nonproprietary Name (INN): Trastuzumab.

[0144] The inhibitor of programmed death ligand 1 (PD-L1) may be an antibody specifically binding to PD-L1 (anti-PD-L1 antibody).

[0145] Exemplary anti-PD-L1 antibodies are disclosed in WO 2010 / 077634 which is incorporated herein in its entirety. Corresponding exemplary anti-PD-L1 antibodies to be used in accordance with the present invention are described below.

[0146] The anti-PD-L1 antibody may comprise a heavy chain variable region polypeptide comprising an HVR-H1, HVR-H2 and HVR-H3 sequence, wherein:(SEQ ID NO: 1)(a) the HVR-H1 sequence is GFTFSX1SWIEI;(SEQ ID NO: 2)(b) the HVR-H2 sequence is AWIX2PYGGSX3YYADSVKG;(SEQ ID NO: 3)(c) the HVR-H3 sequence is RHWPGGFDY;further wherein: X1 is D or G; X2 is S or L; X3 is T or S. X1 may be D; X2 may be S and X3 may be T.

[0147] The polypeptide may further comprise variable region heavy chain framework sequences juxtaposed between the HVRs according to the formula: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4). The framework sequences may be derived from human consensus framework sequences. The framework sequences may be VH subgroup III consensus framework. One or more of the framework sequences may be the following:(SEQ ID NO: 4)HC-FR1 is EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO: 5)HC-FR2 is WVRQAPGKGLEWV(SEQ ID NO: 6)HC-FR3 is RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR(SEQ ID NO: 7)HC-FR4 is WGQGTLVTVSA.

[0148] The heavy chain polypeptide may be in combination with a variable region light chain comprising an HVR-L1, HVR-L2 and HVR-L3, wherein:(a) the HVR-L1 sequence is(SEQ ID NOs: 8)RASQX4X5X6TX7X8A;(b) the HVR-L2 sequence is(SEQ ID NOs: 9)SASX9LX10S,;and(c) the HVR-L3 sequence is(SEQ ID NOs: 10)QQX11X12X13X14PX15T;further wherein: X4 is D or V; X5 is V or I; X6 is S or N; X7 is A or F; X8 is V or L; X9 is F or T; X10 is Y or A; X11 is Y, G, F, or S; X12 is L, Y, F or W; X13 is Y, N, A, T, G, F or I; X14 is H, V, P, T or I; X15 is A, W, R, P or T.

[0149] X4 may be D; X5 may be V; X6 may be S; X7 may be A; X8 may be V; X9 may be F; X10 may be Y; X11 may be Y; X12 may be L; X13 may be Y; X14 may be H; X15 may be A.

[0150] The polypeptide may further comprise variable region light chain framework sequences juxtaposed between the HVRs according to the formula: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). The framework sequences may be derived from human consensus framework sequences. The framework sequences may be VL kappa I consensus framework. One or more of the framework sequences may be the following:LC-FR1 is(SEQ ID NO: 11)DIQMTQSPSSLSASVGDRVTITC;LC-FR2 is(SEQ ID NO: 12)WYQQKPGKAPKLLIY;LC-FR3 is(SEQ ID NO: 13)GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC;LC-FR4 is(SEQ ID NO: 14)FGQGTKVEIKR.

[0151] The anti-PD-L1 antibody (or an antigen binding fragment thereof) may comprise a heavy chain and a light chain variable region sequence, wherein:(a) the heavy chain comprises an HVR-H1, HVR-H2and HVR-H3, wherein further:(SEQ ID NO: 1)(i) the HVR-H1 sequence is GFTFSX1SWIH;(SEQ ID NO: 2)(ii) the HVR-H2 sequence is AWIX2PYGGSX3YYADSVKG;(SEQ ID NO: 3)(iii) the HVR-H3 sequence is RHWPGGFDY, and(b) the light chain comprises an HVR-L1, HVR-L2and HVR-L3, wherein further:(SEQ ID NOs: 8)(iv) the HVR-L1 sequence is RASQX4X5X6TX7X8A;(SEQ ID NOs: 9)(v) the HVR-L2 sequence is SASX9LX10S;(SEQ ID NOs: 10)(vi) the HVR-L3 sequence is QQX11X12X13X14PX15T;wherein: X1 is D or G; X2 is S or L; X3 is T or S; X4 may be D or V; X5 may be V or I; X6 may be S or N; X7 may be A or F; X8 may be V or L; X9 may be F or T; X10 may be Y or A; X11 may be Y, G, F, or S; X12 may be L, Y, F or W; X13 may be Y, N, A, T, G, F or I; X14 may be H, V, P, T or I; X15 may be A, W, R, P or T.

[0153] X1 may be D; X2 may be S and X3 may be T. Furthermore, the positions may be as follows: X4=D, X5=V, X6=S, X7=A and X8=V, X9=F, and X10=Y, X11=Y, X12=L, X13=Y, X14=H and / or X15=A. Furthermore, the positions may be as follows: X1=D, X2=S and X3=T, X4=D, X5=V, X6=S, X7=A and X8=V, X9=F, and X10=Y, X11=Y, X12=L, X13=Y, X14=H and X15=A.

[0154] The antibody (an antigen binding fragment thereof) may further comprise

[0155] (a) variable region heavy chain framework sequences juxtaposed between the HVRs according to the formula: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and

[0156] (b) variable region light chain framework sequences juxtaposed between the HVRs according to the formula: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). The framework sequences may be derived from human consensus framework sequences.

[0157] The variable region heavy chain framework sequences may be VH subgroup III consensus framework. One or more of the framework sequences may be the following:(SEQ ID NO: 4)HC-FR1 is EVQLVESGGGLVQPGGSLRLSCAAS;(SEQ ID NO: 5)HC-FR2 is WVRQAPGKGLEWV;(SEQ ID NO: 6)HC-FR3 is RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR;(SEQ ID NO: 7)HC-FR4 is WGQGTLVTVSA.

[0158] The variable region light chain framework sequences may be VL kappa I consensus framework. One or more of the framework sequences may be the following:(SEQ ID NO: 11)LC-FR1 is DIQMTQSPSSLSASVGDRVTITC;(SEQ ID NO: 12)LC-FR2 is WYQQKPGKAPKLLIY;(SEQ ID NO: 13)LC-FR3 is GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC,;and;(SEQ ID NO: 14)LC-FR4 is FGQGTKVEIKR.

[0159] The antibody or antigen binding fragment thereof) may be or may comprise(a) the variable heavy chain framework sequencesare the following:(SEQ ID NO: 4)(i) HC-FR1 is EVQLVESGGGLVQPGGSLRLSCAAS;(SEQ ID NO: 5)(ii) HC-FR2 is WVRQAPGKGLEWV;(SEQ ID NO: 6)(iii) HC-FR3 is RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR;(SEQ ID NO: 7)(iv) HC-FR4 is WGQGTLVTVSA;;and(b) the variable light chain framework sequencesare the following:(SEQ ID NO: 11)(i) LC-FR1 is DIQMTQSPSSLSASVGDRVTITC;(SEQ ID NO: 12)(ii) LC-FR2 is WYQQKPGKAPKLLIY;(SEQ ID NO: 13)(iii) LC-FR3 is GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC;(SEQ ID NO: 14)(iv) LC-FR4 is FGQGTKVEIKR.

[0160] The antibody (or fragment thereof) may further comprise a human constant region. The constant region may selected from the group consisting of IgG1, IgG2, IgG3 and IgG4. The constant region may be IgG1. The antibody (or fragment thereof) may further comprise murine constant region. The constant region may be selected from the group consisting of IgG1, IgG2A, IgG2B and IgG3. The constant region may be IgG2A.

[0161] The antibody (or fragment thereof) may have reduced or minimal effector function. The minimal effector function may result from an effector-less Fc mutation. The effector-less Fc mutation may be N297A. The effector-less Fc mutation may be D265A / N297A. The minimal effector function may result from aglycosylation.

[0162] The antibody (or fragment thereof) may comprise a heavy chain and a light chain variable region sequence, wherein:

[0163] (a) the heavy chain comprises an HVR-H1, HVR-H2 and an HVR-H3, having at least 85% overall sequence identity to GFTFSDSWIH (SEQ ID NO:15), AWISPYGGSTYYADSVKG (SEQ ID NO:16) and RHWPGGFDY (SEQ ID NO:3), respectively, and

[0164] (b) the light chain comprises an HVR-L1, HVR-L2 and an HVR-L3, having at least 85% overall sequence identity to RASQDVSTAVA (SEQ ID NO:17), SASFLYS (SEQ ID NO:18) and QQYLYHPAT (SEQ ID NO:19), respectively.

[0165] The sequence identity may be at least 90%.

[0166] The antibody (or fragment thereof) may further comprise:

[0167] (a) variable region heavy chain (VH) framework sequences juxtaposed between the HVRs according to the formula: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and

[0168] (b) variable region light chain (VL) framework sequences juxtaposed between the HVRs according to the formula: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4).

[0169] The antibody (or fragment thereof) may further comprise a VH and VL framework region derived from a human consensus sequence. The VH framework sequence may be derived from a Kabat subgroup I, II, or III sequence. The VH framework sequence may be a Kabat subgroup III consensus framework sequence. The VH framework sequences may be the following:(SEQ ID NO: 4)HC-FR1 is EVQLVESGGGLVQPGGSLRLSCAAS;(SEQ ID NO: 5)HC-FR2 is WVRQAPGKGLEWV;(SEQ ID NO: 6)HC-FR3 is RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR;(SEQ ID NO: 7)HC-FR4 is WGQGTLVTVSA.

[0170] The VL framework sequence may be derived from a Kabat kappa I, II, III or IV subgroup sequence. The VL framework sequence may be a Kabat kappa I consensus framework sequence.

[0171] The VL framework sequences may be the following:(SEQ ID NO: 11)LC-FR1 is DIQMTQSPSSLSASVGDRVTITC;(SEQ ID NO: 12)LC-FR2 is WYQQKPGKAPKLLIY;(SEQ ID NO: 13)LC-FR3 is GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC;(SEQ ID NO: 14)LC-FR4 is FGQGTKVEIKR.

[0172] The antibody (or fragment thereof) may comprise a heavy chain and a light chain variable region sequence, wherein:(a) the heavy chain sequence has at least 85%sequence identity to the heavy chain sequence:(SEQ ID NO: 20)EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSA,and(b) the light chain sequence has at least 85%sequence identity to the light chain sequence:(SEQ ID NO: 21)DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR.

[0173] The sequence identity may be at least 90%.

[0174] The antibody (or fragment thereof) may comprise a heavy chain and light chain variable region sequence, wherein:(a) the heavy chain comprises the sequence:(SEQ ID NO: 20)EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSA,and(b) the light chain comprises the sequence:(SEQ ID NO: 21)DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR.

[0175] Moreover, the anti-PD-L1 antibody may be encoded by a nucleic acid. Accordingly, herein described is an isolated nucleic acid encoding the above polypeptide / antibody (or fragment thereof).

[0176] Provided herein is an isolated nucleic acid encoding a light chain or a heavy chain variable sequence of an anti-PD-L1 antibody or antigen binding fragment, wherein:

[0177] (a) the heavy chain further comprises and HVR-H1, HVR-H2 and an HVR-H3 sequence having at least 85% sequence identity to GFTFSDSWIH (SEQ ID NO:15), AWISPYGGSTYYADSVKG (SEQ ID NO:16) and RHWPGGFDY (SEQ ID NO:3), respectively, or

[0178] (b) the light chain further comprises an HVR-L1, HVR-L2 and an HVR-L3 sequence having at least 85% sequence identity to RASQDVSTAVA (SEQ ID NO:17), SASFLYS (SEQ ID NO:18) and QQYLYHPAT (SEQ ID NO:19), respectively.

[0179] The sequence identity may be 90%. The anti-PD-L1 antibody may further comprise a VL and a VH framework region derived from a human consensus sequence. The VH sequence may be derived from a Kabat subgroup I, II, or III sequence. The VL sequence may be derived from a Kabat kappa I, II, III or IV subgroup sequence. The anti-PD-L1 antibody may comprise a constant region derived from a murine antibody. The anti-PD-L1 antibody may comprise a constant region derived from a human antibody. The constant region may be IgG1. The antibody encoded by the nucleic acid may have reduced or minimal effector function. The minimal effector function may result from an effector-less Fc mutation. The effector-less Fc mutation may be N297A.

[0180] Further provided herein is a vector comprising the nucleic acid, a host cell comprising the vector. The host cell may be eukaryotic. The host cell may be mammalian. The host cell may be a Chinese Hamster Ovary (CHO) cell. The host cell may be prokaryotic. The host cell may be E. coli. Also provided herein is a process for making an anti-PD-L1 antibody comprising culturing the above host cell under conditions suitable for the expression of the vector encoding the anti-PD-L1 antibody or antigen binding fragment, and recovering the antibody or fragment.

[0181] The following describes in more detail the herein provided means and methods for treating a cancer and / or a cancer patient.

[0182] Herein contemplated is, accordingly, a pharmaceutical composition comprising a modulator of the HER2 / neu (ErbB2) signaling pathway (like Trastuzumab), and an inhibitor of programmed death ligand 1 (PD-L1) (like the anti-PD-L1 antibody described herein) for use in the treatment of cancer, whereby said cancer is determined to have a low or absent ER expression level and to have an increased expression level of programmed death ligand 1 (PD-L1) in comparison to a control. The cancer may be determined to have a decreased expression level of interferon-gamma (IFNγ) in comparison to the control. The pharmaceutical composition may further comprise a chemotherapeutic agent (like taxol or a taxol derivative, such as dodetaxel (Taxotere®)).

[0183] In accordance with the above, the present invention provides a method for treating cancer comprising administering an effective amount of a modulator of the HER2 / neu (ErbB2) signaling pathway, a chemotherapeutic agent and an inhibitor of programmed death ligand 1 (PD-L1) to a subject in need thereof. The cancer may be determined to have a decreased expression level of interferon-gamma (IFNγ) in comparison to the control.

[0184] Herein provided is a modulator of the HER2 / neu (ErbB2) signaling pathway, and an inhibitor of programmed death ligand 1 (PD-L1) for use in the treatment of cancer, whereby said cancer is determined to have a low or absent ER expression level and to have an increased expression level of programmed death ligand 1 (PD-L1) in comparison to a control. Moreover, herein provided is a modulator of the HER2 / neu (ErbB2) signaling pathway, an inhibitor of programmed death ligand 1 (PD-L1) and a chemotherapeutic agent (like taxol or a taxol derivative, such as dodetaxel (Taxotere®)) for use in the treatment of cancer, whereby said cancer is determined to have a low or absent ER expression level and to have an increased expression level of programmed death ligand 1 (PD-L1) in comparison to a control. The cancer may be determined to have a decreased expression level of interferon-gamma (IFNγ) in comparison to the control.

[0185] As discussed above, the present invention provides a method of treating a cancer in a cancer patient for whom therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated, the method comprising selecting a cancer patient whose cancer is determined to have a low or absent ER expression level and to have an increased expression level of programmed death ligand 1 (PD-L1) in comparison to a control, and administering to the patient an effective amount of a modulator of the HER2 / neu (ErbB2) signaling pathway, of a chemotherapeutic agent and of a programmed death ligand 1 (PD-L1) inhibitor. Likewise, the present invention provides a method of treating a cancer in a cancer patient who is undergoing therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent, the method comprising selecting a cancer patient whose cancer is determined to have a low or absent ER expression level and to have an increased expression level of programmed death ligand 1 (PD-L1) in comparison to a control, and administering to the patient an effective amount of a programmed death ligand 1 (PD-L1) inhibitor.

[0186] The explanations and definitions given herein above in relation to “cancer”, “cancer patient”, “PD-L1 inhibitor”, “PD-L1 inhibitor therapy”, “modulator of the HER2 / neu (ErbB2) signaling pathway”, “chemotherapeutic agent”, “low or absent ER expression level”“increased expression level of programmed death ligand 1 (PD-L1)”, “decreased expression level of interferon-gamma (IFN-γ) and the like apply, mutatis mutandis, in the context of the herein.

[0187] The terms “treatment”, “treating” and the like are used herein to generally mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of partially or completely curing a disease and / or adverse effect attributed to the disease. The term “treatment” as used herein covers any treatment of a disease in a patient and includes: (a) preventing a disease related in a patient which may be predisposed to the disease; (b) inhibiting the disease, i.e. arresting its development; or (c) relieving the disease, i.e. causing regression of the disease.

[0188] A “patient” for the purposes of the present invention includes both humans and other animals, particularly mammals, and other organisms. Thus, the methods are applicable to both human therapy and veterinary applications. Preferably, the patient is human.

[0189] The below explanations relate in more detail to the treatment / therapy of these patients / this patient group in accordance with the present invention.

[0190] The pharmaceutical composition will be formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the individual patient, the site of delivery of the pharmaceutical composition, the method of administration, the scheduling of administration, and other factors known to practitioners. The “effective amount” of the pharmaceutical composition for purposes herein is thus determined by such considerations.

[0191] The skilled person knows that the effective amount of one of the herein described PD-L1 inhibitor(s), modulator(s) of the HER2 / neu (ErbB2) signaling pathway and chemotherapeutic agent(s) in a pharmaceutical composition administered to an individual will, inter alia, depend on the nature of the compound. For example, if said compound is a (poly)peptide or protein the total pharmaceutically effective amount of pharmaceutical composition administered parenterally per dose will be in the range of about 1 μg protein / kg / day to 10 mg protein / kg / day of patient body weight, although, as noted above, this will be subject to therapeutic discretion. More preferably, this dose is at least 0.01 mg protein / kg / day, and most preferably for humans between about 0.01 and 1 mg protein / kg / day.

[0192] The following administration may be employed in respect of Trastuzumab:

[0193] Posology and method of administration

[0194] HER2 testing is mandatory prior to initiation of therapy. Herceptin treatment should only be initiated by a physician experienced in the administration of cytotoxic chemotherapy.MBCThree-Weekly Schedule

[0195] The recommended initial loading dose is 8 mg / kg body weight. The recommended maintenance dose at three-weekly intervals is 6 mg / kg body weight, beginning three weeks after the loading dose.Weekly Schedule

[0196] The recommended initial loading dose of Herceptin is 4 mg / kg body weight. The recommended weekly maintenance dose of Herceptin is 2 mg / kg body weight, beginning one week after the loading dose.Administration in Combination with Paclitaxel or Docetaxel

[0197] In the pivotal trials (H0648g, M77001), paclitaxel or docetaxel was administered the day following the first dose of Herceptin (for dose, see the Summary of Product Characteristics for paclitaxel or docetaxel) and immediately after the subsequent doses of Herceptin if the preceding dose of Herceptin was well tolerated.Administration in Combination with an Aromatase Inhibitor

[0198] In the pivotal trial (B016216) Herceptin and anastrozole were administered from day 1. There were no restrictions on the relative timing of Herceptin and anastrozole at administration (for dose, see the Summary of Product Characteristics for anastrozole or other aromatase inhibitors).EBCThree-Weekly and Weekly Schedule

[0199] As a three-weekly regimen the recommended initial loading dose of Herceptin is 8 mg / kg body weight. The recommended maintenance dose of Herceptin at three-weekly intervals is 6 mg / kg body weight, beginning three weeks after the loading dose.

[0200] As a weekly regimen (initial loading dose of 4 mg / kg followed by 2 mg / kg every week) concomitantly with paclitaxel following chemotherapy with doxorubicin and cyclophosphamide.

[0201] (See section 5.1 for chemotherapy combination dosing).MGCThree-Weekly Schedule

[0202] The recommended initial loading dose is 8 mg / kg body weight. The recommended maintenance dose at three-weekly intervals is 6 mg / kg body weight, beginning three weeks after the loading dose.Breast Cancer (MBC and EBC) and Gastric Cancer (MGC)Duration of Treatment

[0203] Patients with MBC or MGC should be treated with Herceptin until progression of disease. Patients with EBC should be treated with Herceptin for 1 year or until disease recurrence, whatever occurs first.Dose Reduction

[0204] No reductions in the dose of Herceptin were made during clinical trials. Patients may continue therapy during periods of reversible, chemotherapy-induced myelosuppression but they should be monitored carefully for complications of neutropenia during this time. Refer to the Summary of Product Characteristics for paclitaxel, docetaxel or aromatase inhibitor for information on dose reduction or delays.Missed Doses

[0205] If the patient misses a dose of Herceptin by one week or less, then the usual maintenance dose (weekly regimen: 2 mg / kg; three-weekly regimen: 6 mg / kg) should be given as soon as possible. Do not wait until the next planned cycle. Subsequent maintenance doses (weekly regimen: 2 mg / kg; three-weekly regimen: 6 mg / kg respectively) should then be given according to the previous schedule.

[0206] If the patient misses a dose of Herceptin by more than one week, a re-loading dose of Herceptin should be given over approximately 90 minutes (weekly regimen: 4 mg / kg; three-weekly regimen: 8 mg / kg). Subsequent Herceptin maintenance doses (weekly regimen: 2 mg / kg; three-weekly regimen 6 mg / kg respectively) should then be given (weekly regimen: every week; three-weekly regimen every 3 weeks) from that point.Special Patient Populations

[0207] Clinical data show that the disposition of Herceptin is not altered based on age or serum creatinine In clinical trials, elderly patients did not receive reduced doses of Herceptin. Dedicated pharmacokinetic studies in the elderly and those with renal or hepatic impairment have not been carried out. However, in a population pharmacokinetic analysis, age and renal impairment were not shown to affect trastuzumab disposition.Method of Administration

[0208] Herceptin loading dose should be administered as a 90-minute intravenous infusion. Do not administer as an intravenous push or bolus. Herceptin intravenous infusion should be administered by a health-care provider prepared to manage anaphylaxis and an emergency kit should be available. Patients should be observed for at least six hours after the start of the first infusion and for two hours after the start of the subsequent infusions for symptoms like fever and chills or other infusion-related symptoms (see sections 4.4 and 4.8). Interruption or slowing the rate of the infusion may help control such symptoms. The infusion may be resumed when symptoms abate.

[0209] If the initial loading dose was well tolerated, the subsequent doses can be administered as a 30-minute infusion. Pharmaceutical compositions of the invention may be administered parenterally.

[0210] Pharmaceutical compositions of the invention preferably comprise a pharmaceutically acceptable carrier. By “pharmaceutically acceptable carrier” is meant a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The term “parenteral” as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion. The administration of the herein provided compositions may, inter alia, comprise an administration twice daily, every day, every other day, every third day, every fourth day, every fifth day, once a week, once every second week, once every third week, once every month, etc.

[0211] The pharmaceutical composition is also suitably administered by sustained release systems. Suitable examples of sustained-release compositions include semi-permeable polymer matrices in the form of shaped articles, e.g., films, or mirocapsules. Sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman, U. et al., Biopolymers 22:547-556 (1983)), poly (2-hydroxyethyl methacrylate) (R. Langer et al., J. Biomed. Mater. Res. 15:167-277 (1981), and R. Langer, Chem. Tech. 12:98-105 (1982)), ethylene vinyl acetate (R. Langer et al., Id.) or poly-D-(−)-3-hydroxybutyric acid (EP 133,988). Sustained release pharmaceutical composition also include liposomally entrapped compound. Liposomes containing the pharmaceutical composition are prepared by methods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. (USA) 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. percent cholesterol, the selected proportion being adjusted for the optimal therapy.

[0212] For parenteral administration, the pharmaceutical composition is formulated generally by mixing it at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.

[0213] Generally, the formulations are prepared by contacting the components of the pharmaceutical composition uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product is shaped into the desired formulation. Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Non aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes. The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) (poly)peptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, manose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and / or nonionic surfactants such as polysorbates, poloxamers, or PEG.

[0214] The components of the pharmaceutical composition to be used for therapeutic administration must be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). Therapeutic components of the pharmaceutical composition generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0215] The components of the pharmaceutical composition ordinarily will be stored in unit or multi-dose containers, for example, sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution. As an example of a lyophilized formulation, 10-ml vials are filled with 5 ml of sterile-filtered 1% (w / v) aqueous solution, and the resulting mixture is lyophilized. The infusion solution is prepared by reconstituting the lyophilized compound(s) using bacteriostatic Water-for-Injection.

[0216] The herein provided treatment of cancer comprising a modulator of the HER2 / neu (ErbB2) signaling pathway, an inhibitor of programmed death ligand 1 (PD-L1) and a chemotherapeutic agent (like taxol or a taxol derivative, such as dodetaxel (Taxotere®)) may be performed by way of the simultaneous, sequential or separate administration of the individual components of said treatment. For example, one or more of the modulator(s) of the HER2 / neu (ErbB2) signaling pathway as defined herein (like Trastuzumab) may be administered simultaneously with one or more of the herein defined inhibitor(s) of programmed death ligand 1 (PD-L1) (like the herein provided and described anti-PD-L1 antibodies). Also, sequential administration of the modulator(s) of the HER2 / neu (ErbB2) signaling pathway as defined herein (like Trastuzumab) may be administered simultaneously with one or more of the herein defined inhibitor(s) of programmed death ligand 1 (PD-L1) (like the herein provided and described anti-PD-L1 antibodies) to be used in accordance with the present invention is envisaged herein. The herein defined modulators of the HER2 / neu (ErbB2) signaling pathway as defined herein (like Trastuzumab) and the one or more of the herein defined inhibitor of programmed death ligand 1 (PD-L1) (like the herein provided and described anti-PD-L1 antibodies) may also be administered separately. For example, one or more of the modulator(s) of the HER2 / neu (ErbB2) signaling pathway as defined herein (like Trastuzumab) may be administered in a first step followed by administration in a second step with one or more of the inhibitor(s) of programmed death ligand 1 (PD-L1) (like the herein provided and described anti-PD-L1 antibodies) and vice versa. Likewise, the chemotherapeutic agent may be administered simultaneously, sequentially or separately. Any combination of simultaneous, sequential or separate administration of the modulator(s) of the HER2 / neu (ErbB2) signaling pathway, inhibitor(s) of programmed death ligand 1 (PD-L1) and chemotherapeutic agent(s) (like taxol or a taxol derivative, such as dodetaxel (Taxotere®)) is envisaged herein.

[0217] The herein provided treatment of cancer comprising a modulator of the HER2 / neu (ErbB2) signaling pathway, an inhibitor of programmed death ligand 1 (PD-L1) and a chemotherapeutic agent (like taxol or a taxol derivative, such as dodetaxel (Taxotere®)) can be applied as a sole therapy. It may, however, also be applied with one or more additional therapies (i.e. in a further cotherapy with), for example, conventional therapies like surgery, radiotherapy and / or one or more additional chemotherapeutic agents.

[0218] Surgery may comprise the step of partial or complete tumour resection, prior to, during or after the administration of the herein provided cancer treatment comprising a modulator of the HER2 / neu (ErbB2) signaling pathway, an inhibitor of programmed death ligand 1 (PD-L1) and a chemotherapeutic agent (like taxol or a taxol derivative, such as dodetaxel (Taxotere®)). The herein provided modulator of the HER2 / neu (ErbB2) signaling pathway, inhibitor of programmed death ligand 1 (PD-L1) and chemotherapeutic agent (like taxol or a taxol derivative, such as dodetaxel (Taxotere®)) may be administered in a neoadjuvant or adjuvant setting (in particular neoadjuvant or adjuvant treatment of cancer).

[0219] The modulator of the HER2 / neu (ErbB2) signaling pathway, the chemotherapeutic agent and the inhibitor of programmed death ligand 1 (PD-L1) can be administered in a neoadjuvant setting. The modulator of the HER2 / neu (ErbB2) signaling pathway, the chemotherapeutic agent and the inhibitor of programmed death ligand 1 (PD-L1) can be administered in an adjuvant setting or in a metastatic setting.

[0220] Accordingly, the herein provided modulator of the HER2 / neu (ErbB2) signaling pathway, an inhibitor of programmed death ligand 1 (PD-L1) and a chemotherapeutic agent (like taxol or a taxol derivative, such as dodetaxel (Taxotere®)) may be administered to a patient in need of such a treatment during or after a surgical intervention / resection of the cancerous tissue. Therefore, the present invention is useful in neoadjuvant therapy, i.e. the treatment with the herein provided therapy given to a patient / patient group in need thereof prior to surgery. It is also useful in adjuvant therapy (i.e. after surgery).

[0221] The chemotherapeutic agent to be used herein is preferably a taxane (the term “taxol” is used interchangeably herein with “taxane”) or a taxane derivate (taxol derivative), like dodetaxel (Taxotere®) or paclitaxel. The use of dodetaxel / (Taxotere®) is particularly preferred herein.

[0222] The (additional) chemotherapeutic agent(s) may be one or more of the following exemplary, non-limiting, drugs or agents:

[0223] Cisplatin, Vinorelbin, Carboplatin, Paclitaxel, Gemcitabin, Docetaxel, Bevacizumab, Pemetrexed, Etoposid, Irinotecan, Ifosfamid, Topotecan,

[0224] (an) anti-angiogenic agent(s) like a VEGF blocker (such as bevacizumab / Avastin or sutent (sunitinib malate-SU-11248)), linomide, inhibitors of integrin αvβ3 function, angiostatin, razoxin, thalidomide, and including vascular targeting agents (for example combretastatin phosphate or N-acetylcolchinol-O-phosphate));

[0225] (an) cytostatic agent(s) such as antioestrogens (for example tamoxifen, toremifene, raloxifene, droloxifene, iodoxyfene), progestogens (for example megestrol acetate), aromatase inhibitors (for example anastrozole, letrazole, vorazole, exemestane), antiprogestogens, antiandrogens (for example flutamide, nilutamide, bicalutamide, cyproterone acetate), LHRH agonists and antagonists (for example goserelin acetate, luprolide), inhibitors of testosterone 5α-dihydroreductase (for example finasteride), anti-invasion agents (for example

[0226] metalloproteinase inhibitors like marimastat and inhibitors of urokinase plasminogen activator receptor function) and inhibitors of growth factor function, (such growth factors include for example platelet derived growth factor and hepatocyte growth factor such inhibitors include growth factor antibodies, growth factor receptor antibodies, tyrosine kinase inhibitors and serine / threonine kinase inhibitors);

[0227] biological response modifiers (for example interferon); (an) anti-metabolite agent(s) (for example gemcitabine); (an) anti-hormonal compound(s) such as (an) anti-estrogen(s); antibodies (for example edrecolomab); adjuvant (anti-) hormonal therapy / therapies (i.e. therapy with (an) adjuvant (anti-) hormone drug(s), such as tamoxifen; gene therapy approaches (like antisense therapies); and / or immunotherapy approaches.

[0228] The chemotherapy may also (additionally) include the use of one or more of antiproliferative / antineoplastic drugs and combinations thereof, as used in medical oncology, such as (an) tyrosine kinase inhibitor(s), (a) raf inhibitor(s), (a) ras inhibitor(s), (a) dual tyrosine kinase inhibitor(s), taxol, (an) taxane(s) (like paclitaxel or docetaxel), (an) anthracycline(s), like doxorubicin or epirubicin, aromatase inhibitors (such as anastrozole or letrozole) and / or vinorelbine; cyclophosphamide, methotrexate or fluorouracil (which is also known as 5-FU) can be used in such cotherapy individually or in form of a cotherapy comprising these three drugs (“CMF therapy”), optionally in combination with any of the other herein provided additional therapies. Particular examples of chemotherapeutic agents for use with a combination treatment of the present invention are pemetrexed, raltitrexed, etoposide, vinorelbine, paclitaxel, docetaxel, cisplatin, oxaliplatin, carboplatin, gemcitabine, irinotecan (CPT-1 1), 5-fluorouracil (5-FU, (including capecitabine)), doxorubicin, cyclophosphamide, temozolomide, hydroxyurea, (iii) antiproliferative / antineoplastic drugs and combinations thereof, as used in medical oncology, such as antimetabolites (for example antifolates like methotrexate,

[0229] fluoropyrimidines like 5-fluorouracil, purine and adenosine analogues, cytosine arabinoside); antitumour antibiotics (for example anthracyclines like doxorubicin, daunomycin, epirubicin and idarubicin, mitomycin-C, dactinomycin, mithramycin); platinum derivatives (for example cisplatin, carboplatin); alkylating agents (for example nitrogen mustard, melphalan, chlorambucil, busulphan, cyclophosphamide, ifosfamide, nitrosoureas, thiotepa); antimitotic agents (for example vinca alkaloids like vincristine and taxoids like taxol, taxotere); topoisomerase inhibitors (for example epipodophyllotoxins like etoposide and teniposide, amsacrine, topotecan, and also irinotecan); also enzymes (for example asparaginase); and thymidylate synthase inhibitors (for example raltitrexed); and additional types of chemotherapeutic agents.

[0230] Inhibitors / Modulators / chemotherapeutic agents for use in accordance with the present invention are described herein and refer generally to known and / or commercially available Inhibitors / Modulators / chemotherapeutic. However, the use of inhibitors yet to be generated or known compounds to be tested for their inhibiting activity is envisaged in context of the present invention.

[0231] In a further aspect, the present invention relates to the use of (a) nucleic acid(s) or antibody (antibodies) capable of detecting the expression level of ER, PD-L1 and, optionally, IFNγ for determining a patient's need for PD-L1 inhibitor cotherapy in combination with a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent. The respective explanations of said terms have been given above and apply here mutatis mutandis.

[0232] Preferably, the nucleic acid (e.g. oligonucleotide(s)) is (are) about 15 to 100 nucleotides in length. A person skilled in the art is, based on his general knowledge and the teaching provided herein, easily in the position to identify and / or prepare (a) an oligo- or polynucleotide capable of detecting the expression level of ER, PD-L1 and, optionally, IFNγ. In particular these nucleic acid(s) (e.g. oligo- or polynucleotides) may be used as probe(s) in the methods described herein, for example in the measurement of the expression level. A skilled person will know, for example, computer programs which may be useful for the identification of corresponding probes to be used herein. For example, a nucleic acid encoding estrogen receptor (or a part of the nucleic acid) (e.g. SEQ ID NO: 38), a nucleic acid encoding PD-L1 (or a part of the nucleic acid) (e.g. SEQ ID NO: 42) and, optionally, a nucleic acid encoding IFNγ (or a part of the nucleic acid) (e.g. SEQ ID NO: 44 may be used in this context for identifying specific probes for detecting the expression level of ER, PD-L1 and IFNγ, respectively. Exemplary nucleic acid sequences encoding ER, PD-L1 and IFNγ are available on corresponding databases, such as the NCBI database (world wide web at ncbi.nlm.nih.gov / sites / entrez).

[0233] Furthermore, a composition is provided herein which is a diagnostic composition further comprising, optionally, means for detection / determining / evaluating the expression level of ER, PD-L1 and IFNγ. Such means for detection, are, for example, the above-described nucleotides and / or antibodies. Accordingly, the present invention relates to such means (e.g. such nucleotides and / or antibodies) for the preparation of a diagnostic composition for determining a patient in need of a PD-L1 inhibitor cotherapy.

[0234] In an alternative aspect, the present invention relates to such means for detection (e.g the above-described nucleic acids and / or antibodies and / or the “binding molecules” described below in context of the kit to be used in accordance with the present invention) for use in determining a patient in need of a PD-L1 inhibitor cotherapy. Preferably, the present invention relates to (an) antibody / antibodies for use in determining a patient in need of a PD-L1 inhibitor cotherapy.

[0235] Furthermore, the present invention also relates to a kit useful for carrying out the herein provided methods, the kit comprising (a) nucleic acid or (an) antibody capable of detecting the expression level of ER, PD-L1 and, optionally, IFNγ. Also envisaged herein is the use of the herein described kit for carrying out the herein provided methods. Said kit useful for carrying out the methods and uses described herein may comprise oligonucleotides or polynucleotides capable of determining the expression level of ER, PD-L1 and, optionally, IFNγ. For example, said kit may comprise (a) compound(s) required for specifically measuring the expression level of ER, PD-L1 and, optionally, IFNγ. Moreover, the present invention also relates to the use of (a) compound(s) required for specifically measuring the expression level of ER, PD-L1 and, optionally, IFNγ, for the preparation of a kit for carrying out the methods or uses of this invention. On the basis of the teaching of this invention, the skilled person knows which compound(s) is (are) required for specifically measuring the expression level of ER, PD-L1 and, optionally, IFNγ. For example, such compound(s) may be (a) “binding molecule(s)”. Particularly, such compound(s) may be (a) (nucleotide) probe(s), (a) primer(s) (pair(s)), (an) antibody(ies) and / or (an) aptamer(s) specific for a (gene) product of the ER gene / coding sequence, PD-L1 gene / coding sequence and, optionally, IFNγ / coding sequence. The kit (to be prepared in context) of this invention may be a diagnostic kit.

[0236] The kit (to be prepared in context) of this invention or the methods and uses of the invention may further comprise or be provided with (an) instruction manual(s). For example, said instruction manual(s) may guide the skilled person (how) to determine the (reference / control) expression level of ER, PD-L1 and, optionally, IFNγ. or (how) to determine a patient's need of PD-L1 inhibitor therapy. Particularly, said instruction manual(s) may comprise guidance to use or apply the herein provided methods or uses. The kit (to be prepared in context) of this invention may further comprise substances / chemicals and / or equipment suitable / required for carrying out the methods and uses of this invention. For example, such substances / chemicals and / or equipment are solvents, diluents and / or buffers for stabilizing and / or storing (a) compound(s) required for specifically measuring the expression level of ER, PD-L1 and, optionally, IFNγ.Definitions

[0237] As used herein, the terms “comprising” and “including” or grammatical variants thereof are to be taken as specifying the stated features, integers, steps or components but do not preclude the addition of one or more additional features, integers, steps, components or groups thereof. This term encompasses the terms “consisting of” and “consisting essentially of” Thus, the terms “comprising” / “including” / “having” mean that any further component (or likewise features, integers, steps and the like) can be present.

[0238] The term “consisting of” means that no further component (or likewise features, integers, steps and the like) can be present.

[0239] The term “consisting essentially of” or grammatical variants thereof when used herein are to be taken as specifying the stated features, integers, steps or components but do not preclude the addition of one or more additional features, integers, steps, components or groups thereof but only if the additional features, integers, steps, components or groups thereof do not materially alter the basic and novel characteristics of the claimed composition, device or method. Thus, the term “consisting essentially of” means that specific further components (or likewise features, integers, steps and the like) can be present, namely those not materially affecting the essential characteristics of the composition, device or method. In other words, the term “consisting essentially of” (which can be interchangeably used herein with the term “comprising substantially”), allows the presence of other components in the composition, device or method in addition to the mandatory components (or likewise features, integers, steps and the like), provided that the essential characteristics of the device or method are not materially affected by the presence of other components.

[0240] The term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, biological and biophysical arts.

[0241] As used herein, the term “isolated” refers to a composition that has been removed from its in-vivo location (e.g. aquatic organism or moss). Preferably the isolated compositions of the present invention are substantially free from other substances (e.g., other proteins that do not comprise anti-adhesive effects) that are present in their in-vivo location (i.e. purified or semi-purified).

[0242] As used herein the term “about” refers to +10%.ITEMS

[0243] The present invention also relates to the following items:

[0244] 1. A method of determining the need of a cancer patient for a PD-L1 inhibitor cotherapy,

[0245] (i) wherein therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated for the patient or (ii) wherein the patient is undergoing therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent, said method comprising the steps of

[0246] a) measuring in vitro in a sample from said patient the expression level of Estrogen receptor (ER) and of programmed death ligand 1 (PD-L1),

[0247] b) determining a patient as being in need of a PD-L1 inhibitor cotherapy if a low or absent ER expression level and an expression level of programmed death ligand 1 (PD-L1) that is increased in comparison to a control is measured in step (a).

[0248] 2. A method of treating a cancer in a cancer patient for whom therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated, the method comprising selecting a cancer patient whose cancer is determined to have a low or absent ER expression level and to have an increased expression level of programmed death ligand 1 (PD-L1) in comparison to a control, and administering to the patient an effective amount of a modulator of the HER2 / neu (ErbB2) signaling pathway, of a chemotherapeutic agent and of a programmed death ligand 1 (PD-L1) inhibitor.

[0249] 3. A method of treating a cancer in a cancer patient who is undergoing therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent, the method comprising selecting a cancer patient whose cancer is determined to have a low or absent ER expression level and to have an increased expression level of programmed death ligand 1 (PD-L1) in comparison to a control, and administering to the patient an effective amount of a programmed death ligand 1 (PD-L1) inhibitor.

[0250] 4. A pharmaceutical composition comprising a modulator of the HER2 / neu (ErbB2) signaling pathway, and an inhibitor of programmed death ligand 1 (PD-L1) for use in the treatment of cancer, whereby said cancer is determined to have a low or absent ER expression level and to have an increased expression level of programmed death ligand 1 (PD-L1) in comparison to a control.

[0251] 5. The pharmaceutical composition for use in the treatment of cancer of item 4, further comprising a chemotherapeutic agent.

[0252] 6. The method of any one of items 1 to 3, further comprising measuring in vitro in a sample from said patient the expression level of interferon-gamma (IFNγ) and determining a patient as being in need of a PD-L1 inhibitor cotherapy if an expression level of interferon-gamma (IFNγ) that is decreased in comparison to a control is measured.

[0253] 7. The method of any one of items 1, 2, 3 and 6; or the pharmaceutical composition of item 4 and 5, wherein the ER expression level is ER(−).

[0254] 8. The method of any one of items 1, 2, 3, 6 and 7; or the pharmaceutical composition of any one of item 4, 5 and 7, wherein said modulator of the HER2 / neu (ErbB2) signaling pathway is the HER2 antibody Herceptin / Trastuzumab.

[0255] 9. The method of any one of items 1, 2, 3, 6, 7 and 8; or the pharmaceutical composition of any one of items 5, 7 and 8, wherein said chemotherapeutic agent is taxol or a taxol derivative.

[0256] 10. The method of any one of items 1, 2, 3, 6, 7 and 8 to 9; or the pharmaceutical composition of any one of items 4, 5 and 7 to 9, wherein said inhibitor of programmed death ligand 1 (PD-L1) is an antibody specifically binding to PD-L1 (anti-PD-L1 antibody).

[0257] 11. The method of any one of items 1, 2, 3, and 6 to 10; or the pharmaceutical composition of any one of items 4, 5 and 7 to 10, wherein said cancer is a solid cancer.

[0258] 12. The method of item 11; or the pharmaceutical composition of item 11, wherein said solid cancer is breast cancer or gastric cancer

[0259] 13. The method of any one of items 1, 2, 3, and 6 to 12; or the pharmaceutical composition of any one of items 4, 5 and 7 to 12, wherein the expression level of PD-L1 is the mRNA expression level.

[0260] 14. Use of a nucleic acid or antibody capable of detecting the expression level of ER, PD-L1 and, optionally, IFNγ for determining a patient's need for PD-L1 inhibitor cotherapy in combination with a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent.

[0261] 15. The method of any one of items 1, 2, 3 and 6 to 14; or the pharmaceutical composition of any one of items 4, 5 and 7 to 14, wherein said modulator of the HER2 / neu (ErbB2) signaling pathway, said chemotherapeutic agent and said inhibitor of programmed death ligand 1 (PD-L1) are to be administered in a neoadjuvant setting.

[0262] The present invention is further described by reference to the following non-limiting figures and examples. Unless otherwise indicated, established methods of recombinant gene technology were used as described, for example, in Sambrook, Russell “Molecular Cloning, A Laboratory Manual”, Cold Spring Harbor Laboratory, N.Y. (2001) which is incorporated herein by reference in its entirety.BRIEF DESCRIPTION OF THE DRAWINGS

[0263] FIG. 1 provides a schematic of the HER2 protein structure, and amino acid sequences for Domains I-IV, respectively) of the extracellular domain thereof (SEQ ID NOS. 22-25, respectively, in order of appearance).

[0264] FIGS. 2A and 2B depict alignments of the amino acid sequences of the variable light (VL) (FIG. 2A) and variable heavy (VH) (FIG. 2B) domains of murine monoclonal antibody 2C4 (SEQ ID Nos. 26 and 27, respectively); VL and VH domains of variant 574 / Pertuzumab (SEQ ID Nos. 28 and 29, respectively), and human VL and VH consensus frameworks (hum κ1, light kappa subgroup I; humIII, heavy subgroup III) (SEQ ID Nos. 30 and 31, respectively). Asterisks identify differences between variable domains of Pertuzumab and murine monoclonal antibody 2C4 or between variable domains of Pertuzumab and the human framework. Complementarity Determining Regions (CDRs) are in brackets.

[0265] FIGS. 3A and 3B show the amino acid sequences of Pertuzumab light chain (FIG. 3A; SEQ ID NO: 32) and heavy chain (FIG. 3B; SEQ ID NO: 33). CDRs are shown in bold. Calculated molecular mass of the light chain and heavy chain are 23,526.22 Da and 49,216.56 Da (cysteines in reduced form). The carbohydrate moiety is attached to Asn 299 of the heavy chain.

[0266] FIGS. 4A and 4B show the amino acid sequences of Trastuzumab light chain (FIG. 4A; SEQ ID NO: 34) and heavy chain (FIG. 4B; SEQ ID NO: 35), respectively. Boundaries of the variable light and variable heavy domains are indicated by arrows.

[0267] FIGS. 5A and 5B depict a variant Pertuzumab light chain sequence (FIG. 5A; SEQ ID NO: 36) and a variant Pertuzumab heavy chain sequence (FIG. 5B; SEQ ID NO: 37), respectively.

[0268] FIG. 6: FIGS. 6A and 6B show known mRNA transcripts and position of the relevant AFFYMETRIX probe set target regions for gene CD274. Exons are shown as grey bold rectangles, junction regions are indicated by thin horizontal lines. Probe sets with their sequence mapped against mRNA sequences are shown as black bold rectangles. Provided coordinates are genomic coordinates on chromosome 9.

[0269] FIG. 7: FIGS. 7A and 7B show known mRNA transcripts and position of the relevant AFFYMETRIX probe set target regions for gene IFNG. Exons are shown as grey bold rectangles, junction regions are indicated by thin horizontal lines. Probe sets with their sequence mapped against mRNA sequences are shown as black bold rectangles. Provided coordinates are genomic coordinates on chromosome 12.

[0270] FIG. 8 shows the distribution of the expression of genes IFNG and CD274 in the samples of ER- and ER-30 populations. Symbol types correspond to the final pCR status (solid: pCR achieved, open—pCR not achieved).

[0271] FIG. 9A is a box plot of expression of gene CD274 for ER− responders (pCR=YES) and nonresponders (pCR=NO). On the right the histograms of expression for both categories are provided. FIG. 9B shows a distribution of t-test statistics (HO hypothesis of no difference). The vertical mark indicates the actual value found in the involved sample. The area of the shaded regions corresponds to the alpha level.

[0272] FIG. 10A is a box plot of expression of gene IFNG for ER− responders (pCR=YES) and nonresponders (pCR=NO). On the right the histograms of expression for both categories are provided. FIG. 10B shows the distribution of t-test statistics (HO hypothesis of no difference). The vertical mark indicates the actual value found in the involved sample. The area of the shaded regions corresponds to the alpha level.

[0273] FIG. 11A is a box plot of expression of gene CD274 for ER+ responders (pCR=YES) and nonresponders (pCR=NO). On the right the histograms of expression for both categories are provided. FIG. 11B shows a distribution of t-test statistics (HO hypothesis of no difference). The vertical mark indicates the actual value found in the involved sample. The area of the shaded regions corresponds to the alpha level.

[0274] FIG. 12A is a box plot of expression of gene IFNG for ER+ responders (pCR=YES) and nonresponders (pCR=NO). On the right the histograms of expression for both categories are provided. FIG. 12B shows a distribution of t-test statistics (HO hypothesis of no difference). The vertical mark indicates the actual value found in the involved sample. The area of the shaded regions corresponds to the alpha level.

[0275] FIG. 13 is the receiver operating characteristic of the final logistic regression model for ER-population. Positive level is taken to be the positive response status (pCR=YES).

[0276] FIG. 14 is a LIFT curve of the final logistic regression model for ER− population. Positive level is taken to be the positive response status (pCR=YES). The Y-axis displays the ratio of how rich the portion of the population is in the chosen response level (upper curve corresponds to pCR=YES) compared to the rate of that response level as a whole.

[0277] FIG. 15 is an example of predicted clinical response status for ER− population. Shown is predicted profile of response as controlled by patient age, Cancer type, pN status, and expression of both genes involved. The actual predicted pCR probability (which is equal to 0.443) is given for NO LABC patient around 60 y. old and with expression in both genes around median values.

[0278] FIG. 16 is the receiver operating characteristic of the final logistic regression model for ER+ population. Positive level is taken to be the positive response status (pCR=YES).

[0279] FIG. 17 shows the distribution of age of ER− patients.

[0280] FIG. 18 shows the distribution of age of ER+ patients.

[0281] FIG. 19 shows a decision tree view on expression of IFNG and CD274 genes predicting clinical response in ER patients. The first two splits required to explain pCR are the ones wrt to IFNG and CD274.EXAMPLES

[0282] The Example illustrates the invention.Example 1: Cancer Patients Undergoing HER2 Targeted Therapy and Chemotherapy Benefit from PD-L1 Inhibitor Cotherapy, if the Expression Level of ER is Low or Absent (ER Negative) and if PD-L1 Expression Level is Increased

[0283] Estimation of gene expression was performed with the help of R Bioconductor package ‘affy’, R version 2.15.0. All exploratory analyses and predictive models were made using SAS JMP ver. 10.0

[0284] 48 HER2+, ER+ and 39 HER2+, ER− breast cancer biopsies were obtained from NeoSphere clinical trial. The samples had been taken at diagnosis from patients afterwards treated with Docetaxel and Trastuzumab in a neo-adjuvant setting. The distribution of main clinical covariates at base line, as well as of clinical response (as assessed at the surgery) in the involved population is as follows:ER Negative Samples:Patient Age (see FIG. 17)Quantiles100.0%maximum72 99.5%72 97.5%71.55 90.0%64 75.0%quartile54 50.0%median50.5 25.0%quartile44.25 10.0%39 2.5%34.675 0.5%34 0.0%minimum34LevelCountProbCancer TypeIBC 20.04167LABC220.45833OPERABLE240.50000Total481.00000pT (pathologic staging of Tumor)T2180.37500T3150.31250T4150.31250Total481.00000pN (pathologic staging of nodes)N0120.25000N1360.75000Total481.00000G (Grade)G1 10.02083G2150.31250G3160.33333NA160.33333Total481.00000ER Positive Samples:Patient Age (see FIG. 18)Quantiles100.0%maximum74 99.5%74 97.5%74 90.0%65 75.0%quartile57 50.0%median50 25.0%quartile43 10.0%40 2.5%32 0.5%32 0.0%minimum32LevelCountProbCancer TypeIBC 50.12821LABC 80.20513OPERABLE260.66667Total391.00000pTT2150.38462T3160.41026T4 80.20513Total391.00000pNN0110.28205N1280.71795Total391.00000GG2130.33333G3100.25641NA160.41026Total391.00000Contingency Analysis of pathological complete response (pCR) By estrogen receptor status (ER)Count Row %pCR = NOpCR = YESER = ER−2756.252143.7548ER = ER+3384.62615.3839602787Gene Expression ProfilingThe tumor biopsy samples were profiled for gene expression on AFFYMETRIX HG-U133Plus 2

[0287] whole Human Genome microarray platform. Roche HighPure RNA extraction, NuGen amplification and standard AFFYMETRIX hybridization and scanning protocols were used. All array scans passed standard AFFYMETRIX QC.

[0288] Robust Multiarray algorithm (RMA) was used for preprocessing of raw signals (Irizarry et al, 2003. available at ncbi.nlm.nih.gov / pubmed / 12925520). All probe sets available for the genes of interest were retrieved as reported below. For gene CD274, when several probe sets were available to represent this gene, the probe set with the probe set with the highest average expression value (defined as an arithmetical average of expression of a given probe set) was selected to represent the gene:

[0289] CD274 (PDL1)

[0290] 223834_at selected for PDL1

[0291] 227458_at

[0292] The selected probe set corresponds to the last exon / 3′UTR of the gene and captures all known RefSEq mRNAs (see FIGS. 6A and 6B)

[0293] IFNG

[0294] 210354_at

[0295] This probe set also represents the last exon / 3′UTR of the gene and captures all known RefSEq mRNAs (see FIGS. 7A and 7B)

[0296] FIG. 8 shows joint distribution of the expression of the above genes in the samples of both ER− and ER− populations. Symbol types correspond to the final pCR status (solid: pCRachieved, open—pCR not achieved).

[0297] More details on distribution of CD274 and IFNG expression across ER and pCR strata can be found in Appendix I.

[0298] For every ER subpopulation, a logistic regression model was constructed that relates expression of the selected genes with clinical response adjusted for patient age, cancer type, and nodal status:Response∼Patient.Age+Cancer.Type+pN+CD⁢274+IFNG1. ER− Population.

[0299] Summarized model output is given below. Odds ratios are (OR) provided per unit change of biomarker value. As the expression values are given on log 2 scale, one unit change would correspond to 2-fold overexpression. For details see Appendix.ER− populationTermOR (95% CI)LR test p-valueCD2745.2 (1.5; 26.7)0.008IFNG0.30 (0.10; 0.74)0.007Patient Age0.24Cancer Type0.91pN0.87

[0300] The final model for predicting probability for a particular patient to respond to the treatment includes expression of CD274 and IFNG and looks like:p⁡(p⁢C⁢R)=-3.7⁢3⁢7+1.607*CD⁢274-1.069*IFNG2. ER+ Population.

[0301] Summarized model output is given below. Odds ratios are (OR) provided per unit change of biomarker value. As the expression values are given on log 2 scale, one unit change would correspond to 2-fold overexpression. For details see Appendix.ER+ populationTermOR (95% CI)LR test p-valueCD2740.93IFNG0.23Patient Age0.34Cancer Type0.39pN0.92

[0302] The role of PDL1 expression is evident in ER− subpopulation of HER2+ breast cancer patients that underwent combinational treatment with Trastuzumab and chemotherapy in the neoadjuvant setting. Namely, overexpression of PDL1 at diagnosis corresponds to a lower rate of response to neoadjuvant therapy (i.e. a lower rate of response to combinational treatment with Trastuzumab and chemotherapy). This holds irrespective of patient age, cancer type, or lymph node status. A baseline assessment of gene expression of either of the two biomarkers, PDL1 and INFG, respectively, allows to identify if a patient is likely to experience a greater benefit if a PDL-1 targeted therapy is added to Trastuzumab and chemotherapy.

[0303] The following relates to a cut-off value allowing determining a patient as being in need of a PD-L1 inhibitor cotherapy in accordance with the present invention.

[0304] If a gene expression analysis gives a result for IFNG expression higher or equal to 4.8 no combination treatment (HER2-targeted and PDL1-targeted) is recommended and no further PDL1 assessment would be necessary. If a gene expression analysis gives a result for IFNG lower than 4.8 a parallel assessment of PDL-1 is necessary. If PDL-1 gene expression analysis then gives a result of higher or equal to 5.3 a combination treatment (HER2-targeted and PDL1-targeted) is recommended (see FIG. 19).APPENDIX IER− SubpopulationOneway Analysis of CD274 Expression by pCR ER=ERneg (See FIG. 9a)t Test

[0306] YES-NO

[0307] Assuming unequal variancesDifference−0.32948t Ratio−1.94171Std Err Dif0.16969DF45.11513Upper CL Dif0.01226Prob > |t|0.0584Lower CL Dif−0.67122Prob > t0.9708Confidence0.95Prob < t0.0292*

[0308] The results are also shown in FIG. 9B.Oneway Analysis of IFNG Expression by pCR ER=ERneg

[0309] The results are shown in FIG. 10A.

[0310] t Test

[0311] YES-NO

[0312] Assuming unequal variancesDifference0.58405t Ratio2.044225Std Err Dif0.28571DF30.21429Upper CL Dif1.16737Prob > |t|0.0497*Lower CL Dif0.00073Prob > t0.0249*Confidence0.95Prob < t0.9751

[0313] The results are shown in FIG. 10B.ER+ SubpopulationOneway Analysis of CD274 Expression by pCR ER=ERpos

[0314] The results are shown in FIG. 11A.

[0315] t Test

[0316] YES-NO

[0317] Assuming unequal variancesDifference0.25169t Ratio0.898709Std Err Dif0.28006DF6.542171Upper CL Dif0.92345Prob > |t|0.4007Lower CL Dif−0.42006Prob > t0.2003Confidence0.95Prob < t0.7997

[0318] The results are shown in FIG. 11B.Oneway Analysis of IFNG Expression by pCR ER=ERpos

[0319] The results are shown in FIG. 12A.

[0320] t Test

[0321] YES-NO

[0322] Assuming unequal variancesDifference0.5931t Ratio1.501336Std Err Dif0.3951DF7.109044Upper CL Dif1.5244Prob > |t|0.1763Lower CL Dif−0.3382Prob > t0.0882Confidence0.95Prob < t0.9118

[0323] The results are shown in FIG. 12B.APPENDIX II

[0324] Nominal Logistic Fit for pCR ER=ERneg

[0325] Converged in Gradient, 5 iterationsWhole Model TestModel−LogLikelihoodDFChiSquareProb > ChiSqDifference6.784783613.569570.0348*Full26.110299Reduced32.895082RSquare (U)0.2063AICc69.0206BIC79.319Observations 48(or Sum Wgts)MeasureTraining DefinitionEntropy RSquare0.2063 1-Loglike(model) / Loglike(0)Generalized RSquare0.3301 (1 − (L(0) / L(model)){circumflex over ( )}(2 / n)) / (1 − L(0){circumflex over ( )}(2 / n))Mean −Log p0.5440 Σ−Log(ρ[j]) / nRMSE0.4278 √Σ(y[j]−ρ[j])2 / nMean Abs Dev0.3665 Σ |y[j]−ρ[j]| / nMisclassification Rate0.2292 Σ (ρ[j]≠ρMax) / nN48 nLack of FitSourceDF−LogLikelihoodChiSquareLack Of Fit4126.11029952.2206Saturated470.000000Prob > ChiSqFitted626.1102990.1125Parameter EstimatesProb >TermEstimateStd ErrorChiSquareChiSqLower 95%Upper 95%Intercept−5.96882554.16326952.060.1517−15.1153291.70408281Patient Age0.049062380.04250451.330.2484−0.03240340.13829525Cancer Type[IBC]−0.09430231.09822890.010.9316−2.54079772.23824618Cancer Type[LABC]−0.15149450.65444240.050.8169−1.50512691.21757158pN[N0]0.081576360.49795740.030.8699−0.89866221.09707358CD274 Expression1.649792220.71947625.260.0218*0.395338333.2836052IFNG Expression−1.18829780.51220235.380.0203*−2.3323039−0.2889168For log odds of NO / YESEffect Likelihood Ratio TestsSourceNparmDFL-R ChiSquareProb > ChiSqPatient Age111.385744460.2391Cancer Type220.197810330.9058pN110.026907040.8697CD274 Expression117.098004330.0077*IFNG Expression117.153877230.0075*Odds RatioFor pCR odds of NO versus YESTests and confidence intervals on odds ratios are likelihood ratio based.Unit Odds RatiosPer unit change in regressorTermOdds RatioLower 95%Upper 95%ReciprocalPatient Age1.0502860.9681161.1483150.9521217CD2745.2058981.48488626.671760.1920898ExpressionIFNG0.304740.0970720.7490743.2814908ExpressionOdds Ratios for Cancer TypeOddsProb >LowerUpperLevel1 / Level2RatioChisq95%95%LABCIBC0.94441250.97220.028298935.902054OPER-IBC1.4050870.84710.035747968.159191ABLEOPER-LABC1.48778950.65680.25187699.0216463ABLEIBCLABC1.05885930.97220.027853635.337072IBCOPER-0.71169970.84710.014671527.973694ABLELABCOPER-0.67213810.65680.11084453.9701934ABLEOdds Ratios for pNProb >LowerUpperLevel1 / Level2Odds RatioChisq95%95%N1N00.84946150.86970.11145366.033483N0N11.17721650.86970.16574178.9723459Receiver Operating Characteristic(see FIG. 13)Using pCR=‘YES’ to be the positive levelAUC0.79718Confusion Matrix

[0335] Actual

[0336] PredictedTrainingNOYESNO225YES615Lift Curve (see FIG. 14)pCRNO

[0339] YES

[0340] Prediction Profiler (see FIG. 15)

[0341] Nominal Logistic Fit for pCR ER=ERpos

[0342] Converged in Gradient, 19 iterationsWhole Model TestModel−LogLikelihoodDFChiSquareProb > ChiSqDifference2.40059764.8011930.5696Full14.343001Reduced16.743598RSquare (U)0.1434AICc46.2989BIC54.3309Observations (or Sum Wgts)39MeasureTraining DefinitionEntropy RSquare0.1434 1-Loglike(model) / Loglike(0)Generalized RSquare0.2010 (1 − (L(0) / L(model)){circumflex over ( )}(2 / n)) / (1 − L(0){circumflex over ( )}(2 / n))Mean −Log p0.3678 Σ−Log(ρ[j]) / nRMSE0.3462 √Σ(y[j]−ρ[j])2 / nMean Abs Dev0.2351 Σ |y[j]−ρ[j]| / nMisclassification Rate0.1795 Σ (ρ[j]≠ρMax) / nN39 nLack Of FitSourceDF−LogLikelihoodChiSquareLack Of Fit3214.34300128.686Saturated380.000000Prob > ChiSqFitted614.3430010.6351Parameter EstimatesProb >TermEstimateStd ErrorChiSquareChiSqLower 95%Upper 95%InterceptUnstable7.203069093597.51070.000.9984−7043.78847058.1945Patient Age0.05781490.06281120.850.3573−0.05604830.19608254CancerUnstable12.00925137195.01390.000.9987−14089.95914113.9773Type[IBC]CancerUnstable−6.58646833597.5070.000.9985−7057.57067044.39766Type[LABC]pN[N0]−0.05428690.55729040.010.9224−1.16983781.117206CD2740.084852710.98591640.010.9314−1.87046982.14104768ExpressionIFNG−0.73346780.61918171.400.2362−2.04769030.45985303ExpressionFor log odds of NO / YESEffect Likelihood Ratio TestsSourceNparmDFL-R ChiSquareProb > ChiSqPatient Age110.925887320.3359Cancer Type221.891402120.3884pN110.009464440.9225CD274 Expression110.007422130.9313IFNG Expression111.456939450.2274Odds RatiosFor pCR odds of NO versus YESTests and confidence intervals on odds ratios are likelihood ratio based.Unit Odds RatiosPer unit change in regressorOddsLowerUpperTermRatio95%95%ReciprocalPatient Age1.0595190.9454931.2166270.9438246CD274 Expression1.0885570.1540518.5083470.9186476IFNG Expression0.4802410.1290331.5838412.0822891Odds Ratios for Cancer TypeLevel1 / Level2Odds RatioProb > ChisqLower 95%Upper 95%LABCIBC8.3942e−90.212805.1523961OPERABLEIBC2.6876e−80.4499020.868673OPERABLELABC3.20171120.31930.299926236.429388IBCLABC1191292510.21280.1940845.IBCOPERABLE372079930.44990.0479187.LABCOPERABLE0.3123330.31930.02745043.3341535Odds Ratios for pNOddsProb >LowerUpperLevel1 / Level2RatioChisq95%95%N1N01.11468720.92250.107055110.377869N0N10.89711260.92250.09635899.3409878Receiver Operating Characteristic(see FIG. 16)Using pCR=‘YES’ to be the positive levelAUC0.77273Confusion MatrixActualPredictedTrainingNOYESNO321YES60The present invention refers to the following nucleotide and amino acid sequences: The sequences provided herein are, inter alia, available in the NCBI database and disclosed in WO 2010 / 077634 and can be retrieved from world wide web at ncbi.nlm.nih.gov / sites / entrez?db=gene; Theses sequences also relate to annotated and modified sequences. The present invention also provides techniques and methods wherein homologous sequences, and variants of the concise sequences provided herein are used.

[0354] SEQ ID NOS: 1-21 define the anti-PD-L1 antibody to be used in accordance with the present invention. SEQ ID NOS: 1-21 are shown in the sequence listing.

[0355] SEQ ID No. 22 to 37 show sequences of amino acid sequences for Domains I-IV of the HER2 protein (SEQ ID NO. 22-25, see also FIG. 1) and sequences of anti-HER2-antibodies. (SEQ ID NOS: 26 to 37; see also FIGS. 2A, 2B, 3A, 3B, 4A, 4B, 5A, and 5B).SEQ ID No. 26:Amino acid sequence of the variable

[0357] light (Vr) (FIG. 2A) domain of murine monoclonal antibody 2C4 (SEQ ID NOS: 26 and 27, respectively) as shown in FIGS. 2A and 2B.SEQ ID No. 27:Amino acid sequence of the variable heavy (VH) (FIG. 2B) domain of murine monoclonal antibody 2C4 as shown in FIGS. 2A and 2B.SEQ ID No. 28:Amino acid sequence of the variable light (VL) (FIG. 2A) domain of variant 574 / Pertuzumab as shown in FIGS. 2A and 2B.SEQ ID No. 29:Amino acid sequence of the variable heavy (VH) (FIG. 2B) domain of variant 574 / Pertuzumab as shown in FIGS. 2A and 2B.SEQ ID No. 30:human VL consensus frameworks (hum κ1, light kappa subgroup I; humIII, heavy subgroup III) as shown in FIGS. 2A and 2B.SEQ ID No. 31:human VH consensus frameworks (hum κ1, light kappa subgroup I; humIII, heavy subgroup III) as shown in FIGS. 2A and 2B.SEQ ID No. 32:Amino acid sequences of Pertuzumab light chain as shown in FIG. 3A.SEQ ID No. 33:Amino acid sequences of Pertuzumab heavy chain as shown in FIG. 3B.SEQ ID No. 34:Amino acid sequence of Trastuzumab light chain domain as shown in FIG. 4A Boundaries of the variable light domain are indicated by arrows.SEQ ID No. 35:Amino acid sequence of Trastuzumab heavy chain as shown in FIG. 4B. Boundaries of the variable heavy domain are indicated by arrows.SEQ ID No. 36:Amino acid sequence of variant Pertuzumab light chain sequence (FIG. 5A).SEQ ID No. 37:Amino acid sequence of variant Pertuzumab heavy chain sequence (FIG. 5B).SEQ ID NO. 38:Nucleotide sequence encoding Homo sapiens Progesterone Receptor (PR)NCBI Reference Sequence: NC_000011.9>gi|224589802:c101000544-100900355 Homo sapiens chromosome 11, GRCh37.p10 Primary AssemblySEQ ID No. 39:Amino acid sequence of Homo sapiens Progesterone Receptor (PR)PRGR_HUMAN Length: 933 Dec. 7, 2012 15:10 Type: P Check: 6067.SEQ ID NO. 40:Nucleotide sequence encoding Homo sapiens Estrogen Receptor (ER)(NM_000125.3)SEQ ID NO. 41:Nucleotide sequence encoding Homo sapiens Estrogen Receptor (ER)NCBI Reference Sequence: NC_000006.11>gi|224589818:152011631-152424409 Homo sapiens chromosome 6, GRCh37.p10 Primary AssemblySEQ ID No. 42:Amino acid sequence of Homo sapiens Estrogen Receptor (ER)>ENST00000206249_6SEQ ID No. 43:Nucleotide sequence encoding Homo sapiens programmed death ligand 1 (PD-L1)NCBI Reference Sequence: NC_000009.11>gi|224589821:5450503-5470567 Homo sapiens chromosome 9, GRCh37.p10 Primary AssemblySEQ ID NO. 44Nucleotide sequence encoding Homo sapiens programmed death ligand 1 (PD-L1) (CD274), transcript variant 1, mRNANCBI Reference Sequence: NM_014143.3>gi|292658763|ref|NM_014143.31 Homo sapiens CD274 molecule (CD274), transcript variant 1,mRNASEQ ID No. 45:Amino acid sequence of Homo sapiens programmed death ligand 1 (PD-L1) (programmed cell death 1 ligand 1 isoform a precursor [Homo sapiens])NCBI Reference Sequence: NP_054862.1>gi|76615341ref|NP_054862.1| programmed cell death 1 ligand 1 isoform a precursor [Homo sapiens]SEQ ID No. 46:Nucleotide sequence encoding Homo sapiens programmed death ligand 1 (PD-L1) (CD274), transcript variant 2, mRNANCBI Reference Sequence: NM_001267706.1>gi|3909796381ref|NM_001267706.11 Homo sapiens CD274 molecule (CD274), transcript variant 2, mRNASEQ ID No. 47:Amino acid sequence of Homo sapiens programmed death ligand 1 (PD-L1) (programmed cell death 1 ligand 1 isoform b precursor [Homo sapiens])NCBI Reference Sequence: NP_001254635.1>gi|3909796391ref|NP_001254635.11 programmed cell death 1 ligand 1 isoform b precursor [Homo sapiens]SEQ ID No. 48:Nucleotide sequence encoding Homo sapiens programmed death ligand 1 (PD-L1) (Homo sapiens CD274 molecule (CD274), transcript variant 3, non-coding RNA)NCBI Reference Sequence: NR_052005.1>gi|3909796401ref|NR_052005.1| Homo sapiens CD274 molecule (CD274), transcript variant 3, non-coding RNASEQ ID No. 49:Nucleotide sequence encoding Homo sapiens interferon gamma (Homo sapiens chromosome 12, GRCh37.p10 Primary Assembly)NCBI Reference Sequence: NC_000012.11>gi|224589803:c68553521-68548550 Homo sapiens chromosome 12, GRCh37.p10 Primary AssemblySEQ ID No. 50:Nucleotide sequence encoding Homo sapiens interferon gamma, mRNANCBI Reference Sequence: NM_000619.2

[0405] >gi|56786137|ref|NM_000619.21 Homo sapiens interferon, gamma (IFNG), mRNASEQ ID No. 51:Amino acid sequence of Homo sapiens interferon gamma, interferon gamma precursor [Homo sapiens]

[0407] NCBI Reference Sequence: NP_000610.2

[0408] >gi|56786138|ref|NP_000610.21 interferon gamma precursor [Homo sapiens]

[0409] All references cited herein are fully incorporated by reference. Having now fully described the invention, it will be understood by a person skilled in the art that the invention may be practiced within a wide and equivalent range of conditions, parameters and the like, without affecting the spirit or scope of the invention or any embodiment thereof.SEQUENCE LISTINGThe patent application contains a lengthy sequence listing. A copy of the sequence listing is available in electronic form from the USPTO web site (). An electronic copy of the sequence listing will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3).Sequence total quantity: 51 Current application number: US / 19 / 067,334 SEQ ID NO: 1 moltype = AA length = 10 FEATURE Location / Qualifiers REGION 1..10 note = Description of Artificial Sequence: Synthetic HVR-H1 peptide VARIANT 6 note = Asp or Gly source 1..10 mol_type = protein organism = synthetic construct SEQUENCE: 1 GFTFSXSWIH 10 SEQ ID NO: 2 moltype = AA length = 18 FEATURE Location / Qualifiers REGION 1..18 note = Description of Artificial Sequence: Synthetic HVR-H2 peptide VARIANT 4 note = Ser or Leu VARIANT 10 note = Thr or Ser source 1..18 mol_type = protein organism = synthetic construct SEQUENCE: 2 AWIXPYGGSX YYADSVKG 18 SEQ ID NO: 3 moltype = AA length = 9 FEATURE Location / Qualifiers REGION 1..9 note = Description of Artificial Sequence: Synthetic HVR-H3 peptide source 1..9 mol_type = protein organism = synthetic construct SEQUENCE: 3 RHWPGGFDY 9 SEQ ID NO: 4 moltype = AA length = 25 FEATURE Location / Qualifiers REGION 1..25 note = Description of Artificial Sequence: Synthetic HC-FR1 peptide source 1..25 mol_type = protein organism = synthetic construct SEQUENCE: 4 EVQLVESGGG LVQPGGSLRL SCAAS 25 SEQ ID NO: 5 moltype = AA length = 13 FEATURE Location / Qualifiers REGION 1..13 note = Description of Artificial Sequence: Synthetic HC-FR2 speptide source 1..13 mol_type = protein organism = synthetic construct SEQUENCE: 5 WVRQAPGKGL EWV 13 SEQ ID NO: 6 moltype = AA length = 32 FEATURE Location / Qualifiers REGION 1..32 note = Description of Artificial Sequence: Synthetic HC-FR3 polypeptide source 1..32 mol_type = protein organism = synthetic construct SEQUENCE: 6 RFTISADTSK NTAYLQMNSL RAEDTAVYYC AR 32 SEQ ID NO: 7 moltype = AA length = 11 FEATURE Location / Qualifiers REGION 1..11 note = Description of Artificial Sequence: Synthetic HC-FR4 peptide source 1..11 mol_type = protein organism = synthetic construct SEQUENCE: 7 WGQGTLVTVS A 11 SEQ ID NO: 8 moltype = AA length = 11 FEATURE Location / Qualifiers REGION 1..11 note = Description of Artificial Sequence: Synthetic HVR-L1 peptide VARIANT 5 note = Asp or Val VARIANT 6 note = Val or Ile VARIANT 7 note = Ser or Asn VARIANT 9 note = Ala or Phe VARIANT 10 note = Val or Leu source 1..11 mol_type = protein organism = synthetic construct SEQUENCE: 8 RASQXXXTXX A 11 SEQ ID NO: 9 moltype = AA length = 7 FEATURE Location / Qualifiers REGION 1..7 note = Description of Artificial Sequence: Synthetic HVR-L2 peptide VARIANT 4 note = Phe or Thr VARIANT 6 note = Tyr or Ala source 1..7 mol_type = protein organism = synthetic construct SEQUENCE: 9 SASXLXS 7 SEQ ID NO: 10 moltype = AA length = 9 FEATURE Location / Qualifiers REGION 1..9 note = Description of Artificial Sequence: Synthetic HVR-L3 peptide VARIANT 3 note = Tyr, Gly, Phe or Ser VARIANT 4 note = Leu, Tyr, Phe or Trp VARIANT 5 note = Tyr, Asn, Ala, Thr, Gly, Phe or Ile VARIANT 6 note = His, Val, Pro, Thr or Ile VARIANT 8 note = Ala, Trp, Arg, Pro or Thr source 1..9 mol_type = protein organism = synthetic construct SEQUENCE: 10 QQXXXXPXT 9 SEQ ID NO: 11 moltype = AA length = 23 FEATURE Location / Qualifiers REGION 1..23 note = Description of Artificial Sequence: Synthetic LC-FR1 peptide source 1..23 mol_type = protein organism = synthetic construct SEQUENCE: 11 DIQMTQSPSS LSASVGDRVT ITC 23 SEQ ID NO: 12 moltype = AA length = 15 FEATURE Location / Qualifiers REGION 1..15 note = Description of Artificial Sequence: Synthetic LC-FR2 peptide source 1..15 mol_type = protein organism = synthetic construct SEQUENCE: 12 WYQQKPGKAP KLLIY 15 SEQ ID NO: 13 moltype = AA length = 32 FEATURE Location / Qualifiers REGION 1..32 note = Description of Artificial Sequence: Synthetic LC-FR3 polypeptide source 1..32 mol_type = protein organism = synthetic construct SEQUENCE: 13 GVPSRFSGSG SGTDFTLTIS SLQPEDFATY YC 32 SEQ ID NO: 14 moltype = AA length = 11 FEATURE Location / Qualifiers REGION 1..11 note = Description of Artificial Sequence: Synthetic LC-FR4 peptide source 1..11 mol_type = protein organism = synthetic construct SEQUENCE: 14 FGQGTKVEIK R 11 SEQ ID NO: 15 moltype = AA length = 10 FEATURE Location / Qualifiers REGION 1..10 note = Description of Artificial Sequence: Synthetic heavy chain peptide source 1..10 mol_type = protein organism = synthetic construct SEQUENCE: 15 GFTFSDSWIH 10 SEQ ID NO: 16 moltype = AA length = 18 FEATURE Location / Qualifiers REGION 1..18 note = Description of Artificial Sequence: Synthetic heavy chain peptide source 1..18 mol_type = protein organism = synthetic construct SEQUENCE: 16 AWISPYGGST YYADSVKG 18 SEQ ID NO: 17 moltype = AA length = 11 FEATURE Location / Qualifiers REGION 1..11 note = Description of Artificial Sequence: Synthetic light chain peptide source 1..11 mol_type = protein organism = synthetic construct SEQUENCE: 17 RASQDVSTAV A 11 SEQ ID NO: 18 moltype = AA length = 7 FEATURE Location / Qualifiers REGION 1..7 note = Description of Artificial Sequence: Synthetic light chain peptide source 1..7 mol_type = protein organism = synthetic construct SEQUENCE: 18 SASFLYS 7 SEQ ID NO: 19 moltype = AA length = 9 FEATURE Location / Qualifiers REGION 1..9 note = Description of Artificial Sequence: Synthetic light chain peptide source 1..9 mol_type = protein organism = synthetic construct SEQUENCE: 19 QQYLYHPAT 9 SEQ ID NO: 20 moltype = AA length = 118 FEATURE Location / Qualifiers REGION 1..118 note = Description of Artificial Sequence: Synthetic heavy chain polypeptide source 1..118 mol_type = protein organism = synthetic construct SEQUENCE: 20 EVQLVESGGG LVQPGGSLRL SCAASGFTFS DSWIHWVRQA PGKGLEWVAW ISPYGGSTYY 60 ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARRH WPGGFDYWGQ GTLVTVSA 118 SEQ ID NO: 21 moltype = AA length = 108 FEATURE Location / Qualifiers REGION 1..108 note = Description of Artificial Sequence: Synthetic light chain polypeptide source 1..108 mol_type = protein organism = synthetic construct SEQUENCE: 21 DIQMTQSPSS LSASVGDRVT ITCRASQDVS TAVAWYQQKP GKAPKLLIYS ASFLYSGVPS 60 RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YLYHPATFGQ GTKVEIKR 108 SEQ ID NO: 22 moltype = AA length = 195 FEATURE Location / Qualifiers source 1..195 mol_type = protein organism = Homo sapiens SEQUENCE: 22 TQVCTGTDMK LRLPASPETH LDMLRHLYQG CQVVQGNLEL TYLPTNASLS FLQDIQEVQG 60 YVLIAHNQVR QVPLQRLRIV RGTQLFEDNY ALAVLDNGDP LNNTTPVTGA SPGGLRELQL 120 RSLTEILKGG VLIQRNPQLC YQDTILWKDI FHKNNQLALT LIDTNRSRAC HPCSPMCKGS 180 RCWGESSEDC QSLTR 195 SEQ ID NO: 23 moltype = AA length = 124 FEATURE Location / Qualifiers source 1..124 mol_type = protein organism = Homo sapiens SEQUENCE: 23 TVCAGGCARC KGPLPTDCCH EQCAAGCTGP KHSDCLACLH FNHSGICELH CPALVTYNTD 60 TFESMPNPEG RYTFGASCVT ACPYNYLSTD VGSCTLVCPL HNQEVTAEDG TQRCEKCSKP 120 CARV 124 SEQ ID NO: 24 moltype = AA length = 169 FEATURE Location / Qualifiers source 1..169 mol_type = protein organism = Homo sapiens SEQUENCE: 24 CYGLGMEHLR EVRAVTSANI QEFAGCKKIF GSLAFLPESF DGDPASNTAP LQPEQLQVFE 60 TLEEITGYLY ISAWPDSLPD LSVFQNLQVI RGRILHNGAY SLTLQGLGIS WLGLRSLREL 120 GSGLALIHHN THLCFVHTVP WDQLFRNPHQ ALLHTANRPE DECVGEGLA 169 SEQ ID NO: 25 moltype = AA length = 142 FEATURE Location / Qualifiers source 1..142 mol_type = protein organism = Homo sapiens SEQUENCE: 25 CHQLCARGHC WGPGPTQCVN CSQFLRGQEC VEECRVLQGL PREYVNARHC LPCHPECQPQ 60 NGSVTCFGPE ADQCVACAHY KDPPFCVARC PSGVKPDLSY MPIWKFPDEE GACQPCPINC 120 THSCVDLDDK GCPAEQRASP LT 142 SEQ ID NO: 26 moltype = AA length = 107 FEATURE Location / Qualifiers source 1..107 mol_type = protein organism = Mus musculus SEQUENCE: 26 DTVMTQSHKI MSTSVGDRVS ITCKASQDVS IGVAWYQQRP GQSPKLLIYS ASYRYTGVPD 60 RFTGSGSGTD FTFTISSVQA EDLAVYYCQQ YYIYPYTFGG GTKLEIK 107 SEQ ID NO: 27 moltype = AA length = 119 FEATURE Location / Qualifiers source 1..119 mol_type = protein organism = Mus musculus SEQUENCE: 27 EVQLQQSGPE LVKPGTSVKI SCKASGFTFT DYTMDWVKQS HGKSLEWIGD VNPNSGGSIY 60 NQRFKGKASL TVDRSSRIVY MELRSLTFED TAVYYCARNL GPSFYFDYWG QGTTLTVSS 119 SEQ ID NO: 28 moltype = AA length = 107 FEATURE Location / Qualifiers REGION 1..107 note = Description of Artificial Sequence: Synthetic humanized 574 Variable Light Chain source 1..107 mol_type = protein organism = synthetic construct SEQUENCE: 28 DIQMTQSPSS LSASVGDRVT ITCKASQDVS IGVAWYQQKP GKAPKLLIYS ASYRYTGVPS 60 RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YYIYPYTFGQ GTKVEIK 107 SEQ ID NO: 29 moltype = AA length = 119 FEATURE Location / Qualifiers REGION 1..119 note = Description of Artificial Sequence: Synthetic humanized 574 Variable Heavy Chain source 1..119 mol_type = protein organism = synthetic construct SEQUENCE: 29 EVQLVESGGG LVQPGGSLRL SCAASGFTFT DYTMDWVRQA PGKGLEWVAD VNPNSGGSIY 60 NQRFKGRFTL SVDRSKNTLY LQMNSLRAED TAVYYCARNL GPSFYFDYWG QGTLVTVSS 119 SEQ ID NO: 30 moltype = AA length = 107 FEATURE Location / Qualifiers REGION 1..107 note = Description of Artificial Sequence: Synthetic consensus framework Hum kappa1 Variable Light Chain source 1..107 mol_type = protein organism = synthetic construct SEQUENCE: 30 DIQMTQSPSS LSASVGDRVT ITCRASQSIS NYLAWYQQKP GKAPKLLIYA ASSLESGVPS 60 RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YNSLPWTFGQ GTKVEIK 107 SEQ ID NO: 31 moltype = AA length = 119 FEATURE Location / Qualifiers REGION 1..119 note = Description of Artificial Sequence: Synthetic consensus framework Hum kappa1 Variable Heavy Chain source 1..119 mol_type = protein organism = synthetic construct SEQUENCE: 31 EVQLVESGGG LVQPGGSLRL SCAASGFTFS SYAMSWVRQA PGKGLEWVAV ISGDGGSTYY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGR VGYSLYDYWG QGTLVTVSS 119 SEQ ID NO: 32 moltype = AA length = 214 FEATURE Location / Qualifiers REGION 1..214 note = Description of Artificial Sequence: Synthetic Pertuzumab light chain source 1..214 mol_type = protein organism = synthetic construct SEQUENCE: 32 DIQMTQSPSS LSASVGDRVT ITCKASQDVS IGVAWYQQKP GKAPKLLIYS ASYRYTGVPS 60 RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YYIYPYTFGQ GTKVEIKRTV AAPSVFIFPP 120 SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT 180 LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 214 SEQ ID NO: 33 moltype = AA length = 448 FEATURE Location / Qualifiers REGION 1..448 note = Description of Artificial Sequence: Synthetic Pertuzumab heavy chain source 1..448 mol_type = protein organism = synthetic construct SEQUENCE: 33 EVQLVESGGG LVQPGGSLRL SCAASGFTFT DYTMDWVRQA PGKGLEWVAD VNPNSGGSIY 60 NQRFKGRFTL SVDRSKNTLY LQMNSLRAED TAVYYCARNL GPSFYFDYWG QGTLVTVSSA 120 STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG 180 LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKKVEPK SCDKTHTCPP CPAPELLGGP 240 SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS 300 TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSREEM 360 TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ 420 QGNVFSCSVM HEALHNHYTQ KSLSLSPG 448 SEQ ID NO: 34 moltype = AA length = 214 FEATURE Location / Qualifiers REGION 1..214 note = Description of Artificial Sequence: Synthetic Trastuzumab light chain source 1..214 mol_type = protein organism = synthetic construct SEQUENCE: 34 DIQMTQSPSS LSASVGDRVT ITCRASQDVN TAVAWYQQKP GKAPKLLIYS ASFLYSGVPS 60 RFSGSRSGTD FTLTISSLQP EDFATYYCQQ HYTTPPTFGQ GTKVEIKRTV AAPSVFIFPP 120 SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT 180 LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 214 SEQ ID NO: 35 moltype = AA length = 449 FEATURE Location / Qualifiers REGION 1..449 note = Description of Artificial Sequence: Synthetic Trastuzumab heavy chain source 1..449 mol_type = protein organism = synthetic construct SEQUENCE: 35 EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR IYPTNGYTRY 60 ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRWG GDGFYAMDYW GQGTLVTVSS 120 ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS 180 GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG 240 PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN 300 STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE 360 MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 420 QQGNVFSCSV MHEALHNHYT QKSLSLSPG 449 SEQ ID NO: 36 moltype = AA length = 217 FEATURE Location / Qualifiers REGION 1..217 note = Description of Artificial Sequence: Synthetic variant Pertuzumab light chain sequence source 1..217 mol_type = protein organism = synthetic construct SEQUENCE: 36 VHSDIQMTQS PSSLSASVGD RVTITCKASQ DVSIGVAWYQ QKPGKAPKLL IYSASYRYTG 60 VPSRFSGSGS GTDFTLTISS LQPEDFATYY CQQYYIYPYT FGQGTKVEIK RTVAAPSVFI 120 FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS 180 TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC 217 SEQ ID NO: 37 moltype = AA length = 449 FEATURE Location / Qualifiers REGION 1..449 note = Description of Artificial Sequence: Synthetic variant Pertuzumab heavy chain sequence source 1..449 mol_type = protein organism = synthetic construct SEQUENCE: 37 EVQLVESGGG LVQPGGSLRL SCAASGFTFT DYTMDWVRQA PGKGLEWVAD VNPNSGGSIY 60 NQRFKGRFTL SVDRSKNTLY LQMNSLRAED TAVYYCARNL GPSFYFDYWG QGTLVTVSSA 120 STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG 180 LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKKVEPK SCDKTHTCPP CPAPELLGGP 240 SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS 300 TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSREEM 360 TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ 420 QGNVFSCSVM HEALHNHYTQ KSLSLSPGK 449 SEQ ID NO: 38 moltype = DNA length = 100190 FEATURE Location / Qualifiers source 1..100190 mol_type = unassigned DNA organism = Homo sapiens SEQUENCE: 38 agtccacagc tgtcactaat cggggtaagc cttgttgtat ttgtgcgtgt gggtggcatt 60 ctcaatgaga actagcttca cttgtcattt gagtgaaatc tacaacccga ggcggctagt 120 gctcccgcac tactgggatc tgagatcttc ggagatgact gtcgcccgca gtacggagcc 180 agcagaagtc cgacccttcc tgggaatggg ctgtaccgag aggtccgact agccccaggg 240 ttttagtgag ggggcagtgg aactcagcga gggactgaga gcttcacagc atgcacgagt 300 ttgatgccag agaaaaagtc gggagataaa ggagccgcgt gtcactaaat tgccgtcgca 360 gccgcagcca ctcaagtgcc ggacttgtga gtactctgcg tctccagtcc tcggacagaa 420 gttggagaac tctcttggag aactccccga gttaggagac gagatctcct aacaattact 480 actttttctt gcgctcccca cttgccgctc gctgggacaa acgacagcca cagttcccct 540 gacgacagga tggaggccaa gggcaggagc tgaccagcgc cgccctcccc cgcccccgac 600 ccaggaggtg gagatccctc cggtccagcc acattcaaca cccactttct cctccctctg 660 cccctatatt cccgaaaccc cctcctcctt cccttttccc tcctcctgga gacgggggag 720 gagaaaaggg gagtccagtc gtcatgactg agctgaaggc aaagggtccc cgggctcccc 780 acgtggcggg cggcccgccc tcccccgagg tcggatcccc actgctgtgt cgcccagccg 840 caggtccgtt cccggggagc cagacctcgg acaccttgcc tgaagtttcg gccataccta 900 tctccctgga cgggctactc ttccctcggc cctgccaggg acaggacccc tccgacgaaa 960 agacgcagga ccagcagtcg ctgtcggacg tggagggcgc atattccaga gctgaagcta 1020 caaggggtgc tggaggcagc agttctagtc ccccagaaaa ggacagcgga ctgctggaca 1080 gtgtcttgga cactctgttg gcgccctcag gtcccgggca gagccaaccc agccctcccg 1140 cctgcgaggt caccagctct tggtgcctgt ttggccccga acttcccgaa gatccaccgg 1200 ctgcccccgc cacccagcgg gtgttgtccc cgctcatgag ccggtccggg tgcaaggttg 1260 gagacagctc cgggacggca gctgcccata aagtgctgcc ccggggcctg tcaccagccc 1320 ggcagctgct gctcccggcc tctgagagcc ctcactggtc cggggcccca gtgaagccgt 1380 ctccgcaggc cgctgcggtg gaggttgagg aggaggatgg ctctgagtcc gaggagtctg 1440 cgggtccgct tctgaagggc aaacctcggg ctctgggtgg cgcggcggct ggaggaggag 1500 ccgcggctgt cccgccgggg gcggcagcag gaggcgtcgc cctggtcccc aaggaagatt 1560 cccgcttctc agcgcccagg gtcgccctgg tggagcagga cgcgccgatg gcgcccgggc 1620 gctccccgct ggccaccacg gtgatggatt tcatccacgt gcctatcctg cctctcaatc 1680 acgccttatt ggcagcccgc actcggcagc tgctggaaga cgaaagttac gacggcgggg 1740 ccggggctgc cagcgccttt gccccgccgc ggagttcacc ctgtgcctcg tccaccccgg 1800 tcgctgtagg cgacttcccc gactgcgcgt acccgcccga cgccgagccc aaggacgacg 1860 cgtaccctct ctatagcgac ttccagccgc ccgctctaaa gataaaggag gaggaggaag 1920 gcgcggaggc ctccgcgcgc tccccgcgtt cctaccttgt ggccggtgcc aaccccgcag 1980 ccttcccgga tttcccgttg gggccaccgc ccccgctgcc gccgcgagcg accccatcca 2040 gacccgggga agcggcggtg acggccgcac ccgccagtgc ctcagtctcg tctgcgtcct 2100 cctcggggtc gaccctggag tgcatcctgt acaaagcgga gggcgcgccg ccccagcagg 2160 gcccgttcgc gccgccgccc tgcaaggcgc cgggcgcgag cggctgcctg ctcccgcggg 2220 acggcctgcc ctccacctcc gcctctgccg ccgccgccgg ggcggccccc gcgctctacc 2280 ctgcactcgg cctcaacggg ctcccgcagc tcggctacca ggccgccgtg ctcaaggagg 2340 gcctgccgca ggtctacccg ccctatctca actacctgag gtgagggccc gggacggggc 2400 acgcccagcg cgtccgggag tagcggttcc gttggcggcg gcggccgcca accctcagcc 2460 ccagccccag cgcaccgctg cgctccccgg ggcggccgga gagggtgggc agcgggacac 2520 agcacagggg cagttgcctc ccttcttctt ccctcctctc ctcactcttg gggacacgaa 2580 ggtgggcgca gaatatacta tttttggggc gtgcctccct gaaagctgtt tttttgtttg 2640 ttttttaact ttccgaatct tccagattcc gaagcagaac caaccccgat ttaaaacgtg 2700 cagcgtcaca ctaggtccgc tgtagcccag tggggcagaa agtgcgcggc gagttggggg 2760 ctttatgaaa tgcttctttc ttagaagaag gacgtttacc aggagtgctt gtcttggaga 2820 ggagttaagg caccgttccc ccgggagggg tgggacttga gaggtggccg gccagaaccg 2880 aaagcagcac catcttaggg atttgaacac ttcagtggct cagttttctt aagaatctca 2940 agattaaaat taagttcacg tgggaaatgt ttaaactgtg gatttaaacg cctgtcactg 3000 cattgcaccg ttttcttatt attgcttgct attcactaca atttttttta tatacaggtt 3060 taaaaaacac tactttgcat actgaagtaa tggaatgtaa aaaaagaatg ctctgtttgg 3120 aatcttatgt tgtgaatagg caaaacagtg tcagtgtatt ggacaatact ttaaaatgac 3180 aaacatatac ttgcttaagt aagcaatgat tacagggttg tgttttaaaa actcaaaacc 3240 aaaacattgc aaagtaccat cgaactttta aagccaaacc atatttgttt tgacccagca 3300 tacagacagg aaggacataa catttcattt gtcaaagact aaattgtttc tatataaaga 3360 gttttgtaga aagatttcct tttaaccgac tttaactttc taggacataa tattatacac 3420 taattattgt tcttttatat tggtgctact gatgaatggc taatcatttg caagtatggt 3480 gaatccagtt acggatagtc tattaccaag tttagtttgc atgtctttca agtgtatata 3540 tacagttctg tttttaaaat ctcctttcac cctgttaata ctggtttaag aaacctttag 3600 tattagatag tggtgcactt aaaaataaat ggagtacttt gttttgcatt tcaaggccgg 3660 attcagaagc cagccagagc ccacaataca gcttcgagtc attacctcag aagatttgtt 3720 taatctgtgg ggatgaagca tcaggctgtc attatggtgt ccttacctgt gggagctgta 3780 aggtcttctt taagagggca atggaaggta ggctcctttc ccctgatcct ttattattgg 3840 tttaattgta aatggagacc atctaatatt gtatagattt caattattcc ttgtttctta 3900 taagaaatgg tgatatttcc atataattta aaatatatga tgacatttta acaatatgtt 3960 tttatttatg atactcaaaa tggaatgtgg ttgggtacta taattgcata ctctttgact 4020 aacactttca gtattagaca taagtcataa aaatcttgag gacagtgcta ctattgttcc 4080 ttaactgctt agctttgagg aaacagcttt gttctaatag tacttttata tatctattat 4140 gtaggtatat gtgtatgcag tactttaaaa ttttgattaa aagaaaaatg gtagttgaca 4200 catatgtaca tgtatgcgta tacatatttg tacatacacg aacatatatc acaacatgta 4260 tatgatgcag ttttctacat gatactgtct tttgactaca tgaatattta tgtaatattt 4320 acaaagaagt aattctaaac aaatttttga attctctttt tgttcagtat atttttgtaa 4380 gtgtataaga ggacaggatt aaacagttta aaataaaaaa cctggacatc acagtaacat 4440 aatttctaaa gaagtatttt gctttaagta aaactttcat gtttttaaac tcattgaact 4500 tacatgctta atgatttcag atttacttgc atagtgtttc agattttaac tttcaaagaa 4560 aatatttttg aatttctttc tacctaaagt ctaagcagcc aaacatcttt acatttgaag 4620 ataaaaatac attgaaagat ttcatatttt aataccagca ataaatgtat tctataacta 4680 tgtaaaatga agcttaggat tccctctgga gtgctgagat cacacctaga caaggaacca 4740 aggatctgaa tgttggcttt ttgtttcctg ttctgaggat ttttgtttgt ttgtttgttt 4800 caacagccct ctccttacaa gcagaaaagc aggtagtagg aaatttcact ttaagggagc 4860 tttcaaagga gttcttcata acaaatattt gctttgtata tttttagaac atgatttttt 4920 ctcacaaaag atgaatgtat tttactgatg ttgaacatat tcagctttag ggggtttgat 4980 tgcattttaa actaattgag gcagtgttaa aagtggtact tgagaaaata gggcaactga 5040 tagtggctct tacccattga cattatttat ttacagttac agttggaagt tctttgtgtg 5100 gaaaagtcag ttttccaatg ggtaattgga gttaccattt ttatctgact ttggttctgg 5160 tttcttaaac attgccttct gcattaatat gatttcctct cttcttaaag tctcctaagg 5220 atagtataat tttataagtg aatgactccc ttagaatcct cttaagccca atttgtccta 5280 ccccagctcc ttcttttgaa agataagcaa atctcaaaga cataaatatg agtttccaaa 5340 ggtcacaaaa ctagttagta gctgattgtt agtaaacata ggttaaatat ttttacattg 5400 cagcgcttgt aaatcagaga tgatatgcaa aagtagatat ataaactgtt tgattcacag 5460 aagttatttc ataaagtgca tatatagaac aaagagcacc ctaactaaaa tacaaatgct 5520 ttctcgtcat tttgttagaa tagcatccaa aactgtagac gaagtctttc caaatgtact 5580 cttagaaagc atttgttgga ctccggctgt tggcatggtc ctatagtctt gagtactaga 5640 agtgaagcac ctttatttag cagtaattac aaagagttac ttaagattga tgcagataaa 5700 tcattcatga aactagaaca agattatgaa ctacattagt aagttccttc attcagcaat 5760 ttatgccaaa gatacacttt ccctgacttc acttttctct gccttgagat aaaatgagga 5820 taacagtggc tatttcttag ggttgctata aagattaaat gagctgatac ttgtaaagta 5880 tgtaaaagaa ggcctgacat attatcagtt tccattgaca tttctacttt caaggaactt 5940 gtaatatagt tagggaggta acatatgcac ataaaacatc taaataaaga ttctcagtaa 6000 atgcccaagt aagcaattct gtaatgtata tgagatctgt gtggtttgtg agtttttgta 6060 tttggacaga gcgaggtggt tatgggttga aatatgtata ttcttgaatg atgaagaagt 6120 ctacatggaa gatatgaaca tttgattagt aaaggacaaa taagctttct aggcattgag 6180 gagagcttta agtgtataca gtcaaagaag agtgaagaaa ttaagattac actgactaag 6240 cattgaatgt tcacattagg aaattgagag agttaaagtt tgagaaacta gattgctagt 6300 gtttgggtga atttggagaa tcggtaattt aaggcaagag aatatagaga atgttctagg 6360 agttttcagg aatgagaaag ataagtagaa ggacttatat caggttcaaa atcttcaata 6420 aagcaatgct gcgtgatgag ctggttcaag gtggcgctgt gtgtggatgc caatatggcc 6480 agaagtttaa agtaaacagg caacaatatg gatactaatg ttgccaagga tgagaatgaa 6540 aatgatggca ttataaaatg ctttctctgt gccagtgact atttcagtgc tttgcaggtc 6600 ttaacttatt tagttgttat aatgttggta ttattattat tactcccact ttacaaatga 6660 ggaaactaag acctgtagat gttaagctgt cttaatacgc ttaggaaatg gtaaagtcag 6720 gattcaaatc caggtggtat gaatccagaa tcccggccct tgaccactgt gcttcctttc 6780 tcataatagg aaatgcagtc aaagaaaaac aatagagggt tagaagaaaa gatgtgagcc 6840 aagtgatgaa acatctagga aggtaaaagt gaatcctaaa ggagaatgca agagcagggg 6900 taatgagaag tgtgtgatat aaaaggatgg atcatattaa cttcacattt agggcagcaa 6960 taagaagaca taagcaggag ccaacagcca gtttcttcac ctccctccca tgttaggaga 7020 gggaggtgat aaagcctata aagtcattct ggatgactgg gtttccctgg atatgagaca 7080 gaagagagaa gagataaaga aataagatac cactcaggaa atgggagaaa ggagttggga 7140 aaaatgattt cttttaagct aattgaactg tttaggatat agtattggcc aaaaaccagt 7200 tggttggata gcccattgca ttcctttacc aagacttgta ggtttggagt gaaccatgaa 7260 aggaccagga attgacttaa tgccttccaa agagaggaag taatcatgac ctgccagtcc 7320 tacaaatgca gactactaac ctggtatgat gaaggaaagg actatttctc aatggcttat 7380 ctttgccagt acacagtaac cagcccagtg ctaggaatat actaggcatc cagtagataa 7440 ctgctacatg atccagtcat cataactgat aacgccacac ttttattttt tggatgcttt 7500 actcagtgac agcatttgtt gtgaatacat ttggtgtaat atcattaaac acatgttata 7560 atacaattga aatgtattac ttagaagaca ctaagctaag taggtattga aggattttca 7620 attgtattgc atattatgct cacttttttt tttttttttt tgagacggag tctcgctgtt 7680 gcccatgctg gagtgcagtg gtgtgatctc ggctcactgc aagctccgcc tcccgggttc 7740 atgccattct cctgccttag cctcccgagt agctgggact acgggtgccc cccaccacgc 7800 ctggctaatt ttttgtattt tttagtagag atggggtttc accatggtct caatctccta 7860 acctcatgat ccgcctgcct cggcctccca aagtgctggg attacaggcg tgagccacca 7920 cacctggcct gtcctcattc ttttattcat atattaattg ttcatgaagt aattacctta 7980 attacattag ttcatgtatt tattgagtac ctgccatgcg ccaggcacta tgttaggtac 8040 tggtgaaacc acagtaaaac gagagacctt gctggctgtt gactaaagtt tatagtgtgg 8100 tggtggagag agacatttta cctatttgtg tacacatgac taattgcaca tgtggtaagt 8160 gattcgttta tgcaatgttt ggacaactag agaattgacc cctctgtcag ataatgggga 8220 aagtttttca gacctagaca tcacaataaa tagactgcag aggaaactag acagaaatga 8280 aatactttta tgaataaagt gtttctttca aaagttgggt atacttggtg tcataggcta 8340 gtaatgaaaa ctggtttggt agcatagttc tccttgatac agcatcagaa agagaaagat 8400 tgaacatcca atttttcatc agcaacttct gtgatttatt tgttctatat ttagagctct 8460 gtaatgcttt ctccatcttt tgtagtgcac agatcatcca gcaatctctt ctactataat 8520 ctatttgaat ttgaataata tttgggctct gagaagatat tttgccaata atcttattta 8580 tattcattta atctccaata gagtctgctc tatagcagct ttcacattac cctaaaaata 8640 aatatgtagt aattcactag attttatttc aaaactctat atccaatttc ttttcaagct 8700 gaggtctctc agtttactat ttagcatgga taatgataga ctggttttaa gcaccacttt 8760 acattagggt tcattgaaat ctctactgta ctgaaaaaga aatggttaaa aagatagcat 8820 ttggtgtcat ctgttcatat ttggttattt agagctccca gatttttaat actctttcag 8880 aacatgtact ttaataccac tacaagagcc agaggagaag cagtggtatt agccatgcgt 8940 gctagtgcta ataactgctc ctgttctggc cactgaatca ttgtacttta attactcaag 9000 taacacaaaa atccatctcc tttaaaaaat gaaaatgtta tcaataaggc ttaagtcctc 9060 ttgatcaata cctctaatct gttcccttgc ctaccatctt tctatacctc tacctcagtt 9120 tttcccatgt gtatgtgtgt gtggaatatg ttctctgggg tatgttaata tataaatggt 9180 attatatatc ataatttatg catcatgtta ttttgataca aaaatgactt gaagctctat 9240 tgttcatata tgtagttcca acttgttttt ttaaccactg cttaatattt cataatatga 9300 aaaatcatta tatttaggaa ttactaatta cactattact tgagtaaacc ctttttgtac 9360 atttctcctt tggtacatgt cttgcttctc tttccagaat atattgaagt attttgatac 9420 agttctaaat gaactagatg attagctttc tacctgctgc taagatgatt tgattcacta 9480 atttattcac ttcagataca tgtattgatc ctacatttaa aagttgcttg tgctgggact 9540 gaagatgaaa atgtatgcag accctgtctt tgacatgcac aaaaccaaga agaagaaaac 9600 aaaacagaac aaacctgtgg tacaactagt gaacatgaga ataccttgat acaagttatg 9660 ccagagccgt ggccaacaca ccgtagtatt cagttagaca catggagaag aatggcagtt 9720 tattctgcta acatatcttg atgttgtgca agaggcttat tcatttaata gtcacttaag 9780 tcgccttcac tataacccag gcactgtttt aactgcttca ctggattagc tcaattaatc 9840 cttgtaagag tcctttgagg tggaggttct gttagtatct atattttaca gatgggtgta 9900 aagagaggtt tctactattc aggaggtctg actctaactt ctgtatctta aattcctgct 9960 tctgtgctgt tttcctagac tgttttgtat ttgagcaaat attaattact ttatactttt 10020 tcagataaat acatggtctc ttataggaga ctggttttca aattgtgctc cttaactcag 10080 tcaagttcct tcttctattc agaacagctc tgtatttctt tatttggttt atatttccac 10140 ttgagatttt tattgggaca aaggattctc agccaacctt tttttaaaaa gcactccttt 10200 ataacaggaa acccttagat ctcaatcaag tatttattcg agtgactcct aagaagtttt 10260 ttggctctat ttgctggtat ctttttggca tttgtgcaaa gacgagatga ccatttgtga 10320 tcaagattta aagtccaact gctcactctt ctaacagcat ggccacatta ttagccgaaa 10380 atcacatctg agttatagag cttttgcttt tgtcagaaaa aaaacaaagt aactccaggg 10440 aatcttttat cagtcacttc tttaaaggat tgattgaaat aattgacact taagacagac 10500 actcaaaaat aggcacacta atcatttaca caagggattt ccaaccccta ggatgtggac 10560 tggtaccaca gccagtccat ggtccggtgc gggcccatgg cctgttagga agtgggccac 10620 aaagcaggag gtgagcagtg gccaagcgag aattacagac tgagctccgc ctccctgtca 10680 catcagcagc agccttagat tctcataaga gtgagaaccc tattgtgaat tctgcatgca 10740 agggatctag gttgcgcact tcttatgaga atctaatacc tgatgatctg tggtagaaca 10800 atttcatcca gaaaccatcc cacaccatcc atggaaaaat tgtattccac aaaactggtc 10860 cctgatacca aaaaggttga ggactgctgg tttacatctt ggtttttaaa ctccattgtc 10920 atttaagaac atcagaaagt cacatactct gtatatcttc cctatctacc taatttgagg 10980 agaggccgat gaacacaata tccttcttgt ggctagatgg ccctcatata attaactata 11040 taatgcatac ttcatataat ctatcatcaa gacatttttt tctcctctga tgagatatta 11100 tgctttttaa taagtgatat gtaaatagcc tattgggttt tcctttttat tttaactcct 11160 gcaaaacaag tacattttaa tgctcaattt taaaaaatta acaggttttg agctttattt 11220 ttaactgcta ctacagcgtc ttgtgttgtg tactttatat gacattttaa ggaatcataa 11280 cttttttctt aaagcatagt atttactagt aatcattatt tctttaggaa aaaaatgcaa 11340 atacttttca tgctatttac tcttctataa ccaagtgatt tatttatgag atagaataca 11400 catgtagtga gtatggaccg tgagctcaaa acagtgctgt agcatttgta cgaaatataa 11460 aaattaaagg gtaaaaataa tggttatcat ttattgagtg tttactaaat tcaggtactc 11520 ttctaatgtc tctttgttta ctaactcatt taactcttgc aacaaaccta tgagacaggt 11580 tctatttttc tcattttaca gataagattt gttcagttag tcaacagtag agcagaactc 11640 cactcctgga gtcaatgccc ttaacttcca ctggatattc ctttgcagga aaagatacag 11700 tctttgattt caaggagcct gcagtgtgtg tgtggaagtg agtgattaac agacaacact 11760 gcaaatttag tgtttgatta aattgtgtgt tgcagactta gggtgctata agtgaactag 11820 agaggaaagt accaagagtt gtcattactg attgcctata tataatagaa accatgctaa 11880 atgctttata tttaatattt ctattcttca caacaatcct aaaaagtaga atttaccatt 11940 tccacttcat atgtaagcaa agactcagaa gttagttaac ttaccccaat taatagtagt 12000 actggcagag atgtgtctgc agtagttaga aaaggtagtt tattgagcta gtaagattta 12060 ttcaaagcct ttaaaaacag agacagtaat taagaaacgt ttatttttga tagttttttt 12120 aatataataa tattgtgact cctggcgtat tatacacaag aaaaatttca ggtgggtcaa 12180 caacttaatt ttttttttat ttgaagaaaa gaaacctaaa agtatggaaa gatgatggag 12240 gtatgtattt atagtcttgg gatggaaaac tttctaaatt tgatataaaa tccagaattc 12300 aaaaacaaat ttgctccatt tgctatgtaa tctcaaattt cttatataag gcaaaaaatt 12360 cataaagtca aagacaacaa caaattagga aactgtaaca tatatgtaaa tcaaaaggaa 12420 attattaccc atatacaaaa tattcttaca aatcaagaaa aagacaaaca ggaagaaaaa 12480 tgaacaaaaa atattaagac agttcacaaa gtacagccca cagaagtatg aagaaggctc 12540 aactcactaa taagcattac tgattaaaat aataatggga atttcagcaa tttgagtact 12600 ggaataataa tgaataacaa ataatgacca ttggagaatc tgaagatagt ttggtattgt 12660 caattaaaat atgcgttact ttggcccaac aattctactt ctagaaactc atccttacag 12720 aactctagaa tacctgtagg tggatgttta ttgcagtatt acattattta taatactgag 12780 aaactgaata cagcccaaat gtctgttaaa aagagattag caaattgtgg tatacccttg 12840 caatgaaaag agtgagttag ctgtctatac tacagatgtc aagaagtctc taaaatattt 12900 gattttatga aaaagcaagg aacaatatca ttctgggtaa aaaattgtac ccatttgatc 12960 ttgctaggta tagagagcta tctaaaagaa aataactaag tgcttactgt caggatgaga 13020 gacttccaat ttctgatgac atgttttggt cttttttttt tttttttttt ttttgaggtg 13080 gaatctagct ctgtcaccca gcctggagtg cagtggtttg acctcagctc actgcaacct 13140 gtgcctccct ggttcaagtg attctcctgc ctcagcctcc cgaatagctg ggattacagg 13200 tgcccaccac catgcccagc taattttttt atttttagta gagacagggt ttcaccgtgt 13260 tgaccagatt ggtctcgaac tcctgacctc aggtgatcct cccgccttgg cctcccaaag 13320 tgttgggatt acaggcatga gccactgcac ccagcttcat tctttaagca agtatttact 13380 gaaggggctg caattacaca ccaacttggt gcctgacttt cctccacact ggccccgcct 13440 agtctcagct agcctctgct gccctaatgg tgtccagtga ccatatgtgc cacttttcac 13500 cttcaggtac cctagaatgt tgaaagagac taacaaagaa tgaaacttga agaatattga 13560 agcaaaagct tgaggttact ctgtatcatg aattaaggaa tccttcctga ctagatggag 13620 ccaagaatgg acacaggccc acaattcctc acacacaatc tcaatattca aaaagcactg 13680 caaacaaaaa ttttgttgta gtcacttgtc aacaatacct aacctgacgt gaatttgttt 13740 ggcagcaaaa tctgatctga aatcacatga ggaaatcttg tagtctatgt aaatattcgt 13800 acattttgct gcataaatat aatgttttat taagtgttgc tctattttat ctttctaaaa 13860 tacaaacaat tttgaatttt gaaacacaat tggcccaata catttcagct aagagatttt 13920 ggacatgtac tcagaaatga tttatagact atgttgagag tggtgcctgc tggctttttg 13980 caatgtagca gccctgaaaa tttttataaa caattcttat catttattct tatctttttt 14040 ttttgagagg gagtccccgc tctgtagccc aggctgaagt gcagtggcac aatctcagct 14100 cactgcactc tgcctcctgg gttcaagcga ttctcctgcc tcagcctccc gagtagctgg 14160 gattacaggc atgtaccacc acacacacta atttttgtag ttttagtaga gatggggttt 14220 caccatgttg gccaggttgc tcttgaactc ctgacctgaa gtgatcctcc caccttggca 14280 tcccaaagtg ctgggattac aggcgtgagc aacagcgccc agcctattct tatcattttt 14340 aataaattct tagtaccatg ctgggttcta cagggagcta taaaagataa ataagccata 14400 ttacctaact tcatatagtt ttcgttcaat catggagttt atcccatatt caagtataat 14460 ataggtaata tataaatcaa atgtgttttt gatttcaagg ggaaaaagat catatttagc 14520 tagggagacc aaaatgtgtt tttatggaaa ggatagcatg ctagatgggc tgctgagaat 14580 gcatgaaatt tttgatagca agaattgtaa tggcttttgt aaacaaaaac tacagactga 14640 gcagaaggac aatttggcta aagcatggaa catggtcaga atcccttacg attatatata 14700 aattatttgt gttttttcta ggataattct tttcatgagg ttttgaaaga gatcagtgac 14760 cccttattcc cacccccaaa aaagttagct taccactgct ttaaaggaat ggtgaggatt 14820 attataaaac agaaggtcaa gaaaatgtgg aatgcacaat ggggtgttta caattaattg 14880 agtaaagagt gagaagcccc taaatatttt ttgggcagtg tagtggcata ttttaatatt 14940 taagaaaatt agtatatcag cagtatattg gaaagattaa tgtcagaaag gtaagggaca 15000 aggaaaacag tctggggatg gtgcactaag gacctgagcc agggttgtgg agagaggaat 15060 gggttcaatg ggtagattgg aaccaattga gaagggtgat gaaagcgaca cagagaattg 15120 tgggggtggg ggacgtcacc ttcagaaata ggaagtacat gatcatttta ctcagtaaag 15180 gagtgatgtt gaaagtagta tttaggaagg attatcctaa aagcaccatg cagaatatat 15240 ttgtacagag attagatagc ttgaagtcag agagaacagc taggaaactg gtaacacaga 15300 ttctgagaag tagtttaatt tcaaattgtt tgtattatat tctacataat gtcctcagca 15360 ggatattgaa ataatataat ttttgtgcac aaaataactt atgagaattg agatgttctg 15420 gtttgtcact tcatttagtc gtctgcttat ttattaattt ataccaaaaa taatgccgtt 15480 ttgtgcctgg atttgtgtta aatattgaga ctcaaaaatg gatgagccaa gttcataact 15540 taggagaaag gcacataaac acgttgcaga aatgcagtct ggtaatatct atgagaggtg 15600 tgagctgaga attgtggaag tataggacag gaagatctaa ccctggtgtt taaatgcggg 15660 gtatggaagg agaaagtctg aatcctgact aagtattaga ctaaaaatat taaatcttaa 15720 aaattattgt aatttcaaag tttgtggcaa cttttttgta attaacagtg gagaccacaa 15780 tggtttatct tctttaagca gagcacttca gtgagtgttt ctttgtgcaa aggcccatcc 15840 agaggccact ttggatgggg gggcggatga aagtatgaag aactgaacct ctctatgtac 15900 tattttaata gaaattaggg aattgtaggc tagcagctgg ggcaaggagt aggaaataga 15960 aagggcctaa caactgagta ggaagcaagg gctcaaggag agtagatgcc tgtgagaatt 16020 tccctattgc aggcaacaat ccataaatcc ctaggctggt agtgcctgat agcaaacttg 16080 gaattgttta ctatctcatg ttttaattac ctccagctac ctcccaaccc ttaagtaaat 16140 ttttgctgtt tacattttcc tttataaaag gatatgcatt tcagattgct gacctttcct 16200 atgatgacaa gtgtctcagt agtatgatac ttagaagata tataggccgg gtgcaatggc 16260 tcacgcctgt aatcccagca ctctggaagg ccgaggtggg cagatcacga ggtcaggaga 16320 tcgtgaccat cctggctaac acggtgaaag aaaccccgtc tctactaaaa atacaaaaaa 16380 aaaaaatagc caggcatggt ggtgggtgcc tgtagtccca gctactcagg aggctgaggc 16440 aggagaatgg catgaaccta ggaggtggag cttacagtga gtggagatca caccactgca 16500 ctccagcctg ggtgacagag caagactctg ggaaaaaaaa aaagaagata tttagcagaa 16560 ggtacagtta atctgcccct tcaagttaaa gataatgttc atgatagaaa aaaggaggta 16620 acttctgttt gaaagacact gttgttttgt agatagcatt ttgaaaacaa gtctttgggg 16680 atgattccac tgttatttga ttttgttgtg aaggtattat aagagatcaa aattttatag 16740 tttcatacat aaacattttg aaataagatt ttctaacttg ctaaaaaatc ttccaaaaaa 16800 attcagttag gttctgaaca catttcataa tagtggcatt tattgaacac atatgtcagg 16860 aacttggcta aatgtgttat attaaacaca aagattttaa atcacttgca cagataaata 16920 gtaaataaaa tggtagagct gagattggaa cccacagaac cttctctctt atttgttaat 16980 attacctctt attcattatc tagtgtctct ttgtcctaag tatttgtaat gaaaattgat 17040 tgacatcagg gagagtagat acttgctagc tgaatttctg gggtaaaaga agcttttcat 17100 aactggtatg tggaattgaa agtgaatatt atgactagat aatctgccct gcagacagtc 17160 ccctcattct ccatggcttc tgggatggct gtcttccacc ttctcttctc agcctgccag 17220 cactcccctg tcctcacttt cagctaacag ccttgcttcc taattttcgg aaaaaaactt 17280 tctgaaattg ctaccaccat ctgtatggac agaagtggcc tctgaactat aatcccattt 17340 ttctggggtt accagagctc tgcagttctc aagtccagcc ctctctcctt gaccactaga 17400 tccagattac tcctgctgac tcaaggacag gctctaccaa tcctcccgca acatttaaat 17460 catttttccc cttctctact ggataattcc tgtcagccta caacatgctc ttccttttct 17520 catcttaaaa tataaacaaa ctacttttgt tgatttcact tactcttcag ccaccaatca 17580 tttttcttct tttacagcaa aactcttcta acacattttc ttctcccatt cactcttaaa 17640 cctaacccaa tcagttgttg acctctgtca ctccaccaaa acttctcttg tcaaggttac 17700 catgttacta aatcttatgg gcaattgtca gtcccatgtt acttgactca ttggtagcat 17760 ttgacaattg atcactccac cttcctgcaa atactttatt cgcttggttt tcaagacacg 17820 accctctctt gattttcctc actagctccc cttcaaactg ttcttctcct ttagctttta 17880 atatgagagg gtccagtgct gagtattctg agtatttggt cctcttccct tctgtttata 17940 tacttacttc cttggttatc taatccagtc tccaaactgt ggataaacca cccaaaatcc 18000 aatcacttcc aaatttctat ctccagccca gacctttccc ctgaactcca gactgaatat 18060 ctcaaaccaa cacatcccaa agttggctcc tgatctactt agggttttct ctggtaagcc 18120 agtgttcttt tggtcctcaa gctatctgcc tcagaatcac ctgggtactg gtttgagatg 18180 atttctgcac ctcgccccag gtaagccaag tcaaaatatc taagaaaggg acccagaaat 18240 ggcatttcag ctagctgttt ggttgtttct taaagttctg cttggctcag aggggagcca 18300 gatacaaagc tgagaggtga aatggttggc tttgtttttg agaacattaa ctctagaagc 18360 aatggggaat agaggcagaa ggtcatgcca gggagatcag acctaagatt atttaaaata 18420 ctctttgatc gagatctggg ggccttcagt cttatgctga gaaaacacta cttttcattc 18480 cctaaagcag tgaacctgaa atatagaagg caggcaggct gtggccccta ccaagaatgc 18540 tcctttctct aggaagaatt ctaaaaaatg tgagaggcta gggagggagg ttggagactt 18600 ccactttttg tgtccttact atataccagg cactatagta gacacattat atgtatttgt 18660 tataggcata ccttagagat atcgcaggtt ctgttctaga caactacaat aaagcaaata 18720 ttgcaataaa gcaagtcaca caaatttttt gctttctggt atctaaaaaa gttatgttta 18780 cactatactg cagtctgtta agtgtgcagt agctttatgt ctaaataaac aatgtacata 18840 ccctgattga aaatacttta ttgctaaaat tgctaatgat cacatgagcc ttaagggagt 18900 tgtaatgttt ttgctggtag agggtcttgc cttgatgtga atggtcattg acttatcgag 18960 gtagtgattg ctggaggttg gggtgtctgt ggcaatttct ttaaataaga caacagtgaa 19020 gtttaccata tccatagact gttcctttca caaaagatta ctctgtagta tgcaatgctg 19080 tgttttaccc acagtagagc ttctttgaaa attggagtca cttttctcaa aacctgctgc 19140 tgcactatca cctaagttta tataatatcc taaatcctaa atcttttgtt gtcatttcaa 19200 caatgctcac agcgtcttca ccaggagcag attgaatctc aagaaaccgc tttctttgct 19260 catccatgag acaagacccc tcatgtgttc aaattttatg agattttagc aattcagtca 19320 catctttgaa ctccacttct aattccagtt cttttgctat tcctaccaca tatgcagtta 19380 cttcctccat gaagtcttga atccctcaaa gtcatctata agggtttgaa tcaacttctt 19440 ccaaactgct gttaatgctg atgctttgac tttctccgat taaccacaaa tgttcttaat 19500 gacatctaaa atgatgaacg ctttctaaaa gattttcaat gtactttgcc cagattcatc 19560 agaggaatta caatctatgg cagctgtagc ctaataaaat gagtttctta agtcataagg 19620 cttgaaagtc agaattactc agtgatccat gggctgcaga atagatgttg tgttagcagg 19680 catgaataca ttaaccttct tgtacatttc catcagagct catgggaatt tttttttcta 19740 agcagtaggt ctcaacagtg ggcttaaagt agttagtgga ccatgctgta aatagatgtg 19800 ctgtcactca ggctttgttg ttccatttct agaacacaag cagagtagat ttagcataat 19860 tcttaagggc cttaggaact tcagaatgat aaatgaggat tggctttaac ttcaaatgcc 19920 cagcttcatt agcccctaac aagagagtca gcctttcctt tgaagctttg aagccaggca 19980 ttgacttctc ctatctagct ctgaaagttc taaatggcat ctttttccaa gagaagactg 20040 ttttatctac attgaaaatc tgttgtttag tgtagctacc ttcatccatg ggcttagcta 20100 gatcttttgg ataacttgct gcagcttcta catcagcact tgctgcttca ccttgtactt 20160 ttgtgttatg ggaatggctt ctttccttta acctcataat ccagcttctg ttttcaaact 20220 tttcttttac agctttcatc cctctctcac ccttgataga attgaagaga attatggcct 20280 tgctctggat taggtttggc tgaagggaat gttgtggctg gcgtgatcct ctatccagac 20340 cattaagcgt ttctccatat cagctataag gctttttttg ttttgttttt ttgctttctt 20400 atcattcatg tgttcactgg agtagcactt tttaacttcc ttcaagaaca tttcctttgc 20460 atttacaact tggctaactg gtgcaagagg cctagctttt ggcctgtctt ggcttttgac 20520 attcctccct cacgaagctt aatctttcta gcttttaatt taaggtgaca catatgcaac 20580 ccttcctttc acttgaacat ttagaggcca ttgtagggtt gttaattggc ctgatttcaa 20640 tattgttgtg acacagggaa taggaaatca tgaagaaaga gacaaggaca gaatgccaat 20700 tggtggagca gtctgaacac acacatttat cagttaagtt tattgtctta catgggcaag 20760 gtttgtggca cccaaaaaca attacagtaa taacatcaaa gatctctgat catagatcac 20820 catagcagag atgataatga aaaagtttga aatattttga gaattactaa aacgtgacag 20880 agacaggagg tgaacatgtg ctgttggaaa aatgatgccc atagtcttgc tggatgcagg 20940 gctgccacaa accctgaatt tgtaaaacat acaatatttg tgaagtacaa ttaagcaaag 21000 cgcaataaga tacgatatgc ttgtatctcc cttaattttc agctaagagc tgtttgagaa 21060 gttactgcca tctccttttt tcagatataa gaattaaaac aaggaaggaa gttaactttc 21120 ctaaagatac ctagccagtt agtaatggag ctggtaacca aacccagttc tggcagactc 21180 caaaaattaa accgctttca ctaaagcaca ccactgggca ctgtgtttga agcacgagat 21240 gatattagca actgattgaa gatttattga aaagacttaa aactcttatt gtagtcaagt 21300 cgtttaaaaa tattttttac ttttaaaatt tattattatt tagagacagg ctggagtgca 21360 gtggcatgat catagctcac tgcagccttg aactcctggg ctgaagtgat actcccacct 21420 caacctccag atattttttt aatagacaag gctttgcttt gttgcaggct ggtctggaac 21480 tcctggcctc aagcaatcct cccaaagtgc tggagtttca ggcataaacc accttgccta 21540 ggctcatttt tttttttttt tttttagacg gagtctcatt ctgtctccca ggagtgtaat 21600 ggcacaatct ctgctcactg caacctctgc ctcccaggtt caagtgactc tcctgcctca 21660 gcctccagag tagctgggat tacaggtgcc caccaccaag cccacctaat ttttttattt 21720 ttagtagaga cagggtttca ctgtgttggc taggctggtc tcaaactcct gaccttaagt 21780 gatctgcctg cctcagcctc ccaaagtgct gggattacag gcgtgagcca ctgcacctgg 21840 ccagttcaaa tcttatatat tttggtgcct agacttaaat aacatttagg aaaatgttaa 21900 ttaattatcc atatagttct acctttttct ttcttttttt ttttttttac aaagccagtt 21960 tcaagtaaga ctggggagat tctagaaagt tataacagaa tcttaagtca cttgatatgc 22020 ctaaacagaa tttgaagaaa taatgaagtt gaacacattg ataaacttag gatttctgta 22080 atcaggaaga aaaaactaaa cttcatagat atgagggaag gaattaacca tttgcatgta 22140 tgtaactgtt ataatcctac atcagagtag cacaaggaca tgaacaactt tgttgacaaa 22200 tagttttgta attttttaat gttttttatg tagctgtctt aaactccttt ctcaatagta 22260 tgtgaggtca aatgattgtg gtttcttttt aaaattttgc atgtaatgtg tagttcctta 22320 cattctctaa atgcttactg tttagctaac tatgttatta tggatgtcaa ttataactaa 22380 gtacttcata caatttcacg ttttataagt tagaagatat gtgaataatt agggcatttg 22440 gaagagttcc agagagattt ttttatttag aaagagagga gcagaacagc ttcaggaaag 22500 atagatgaaa agctgattca ttcaatttaa gatagttgaa gggaagaaaa aagaacacat 22560 tatttagaat gatgacatga agaacttaat ttcctttttt tacttatact ttaagttctg 22620 gggtacatgt gcagaacgtg cagttttgtt acatagatat acacgtgcca tggtggtttg 22680 ctgcacccgt caacccgtca cctacattag gtatttctcc taatgctatc tctcccctag 22740 ccccccatcc cctgacaggc ccaggtatgt aatgttcccc tccctaagtc catgtgttct 22800 cattgctcaa ctcccactta tcagtgagac aatgcggtgt ttggttttct gttcttgtgt 22860 tagcttgctg atgatggttt ccagcttcat ccatgtccct gcgaagaaca tgaactcatt 22920 cttttttatg gctgcatagt attccatggt gtatacgtgc cacatttgct taatccagtc 22980 tgtcattgat ggacatttgg cttggttcca agtctttgct attgtgaata gtgcttcagt 23040 aaacatatgt gtgcctgtgt ctttatagta gaatgattta taatcctctg ggtttatacc 23100 cagtaatggg attgctgggt caaatggtaa ttctagttct agatccttga ggaattgctg 23160 cactgtcttc cacaatggtt gaactaattt acactcccac caacagtgta aaagctttcc 23220 tatttctcca catcctctcc agcatctgtt gtttcctgac tttttaatgg ttgccattcc 23280 aactgccatg agatggtatc ttattgtgat tttgatttgc atttctctaa tgactagtga 23340 tgatgagcat tttttcatgt ctgtcggctg cataaatgtc tttttttgag aagtgtctgc 23400 tcatatcctt tgcccaattt atgaggttgt tttttccttg taaatttaag ttccttctag 23460 attctggata ttagtccttt gtcagatgga tagattgcaa aaattttctt ccattctgta 23520 ggttgcctgt tccctctgat gatagtttct tttgctgtgc agaagttctt tagtttaatt 23580 agatcccatt tgtcaatttt ggcttttgtt gccattgctt ttggtgtttt agacatgaag 23640 tctctgccta cacctatctc ctgaatagtg ttgcctaggt tttcttctag gatttttatg 23700 gtttgcagtc ttacatttaa gtctttaatc catcttgagt taatttttgt ataaggtgtt 23760 aggaaggggt ccagtttcag ttttctgctt atggctagcc agttttccca acaccattta 23820 ttaaacaggg aatcctttcc ccattgcttg tttgagtcaa gtttgtcaaa gatcagatgg 23880 ttgtatatgt gtggtgttat ttctgaggtc tcttttctgt ccattggtct atatatccgt 23940 tttggtacca gtgccatgct gttttggtta ctgcagcctt gtagtatagt ttgaagtcag 24000 gtagtgtgat gcctccagct ttgttctttt tgcttaggat tgtcttggct atgtgggctc 24060 ttttttggtt ccatatgaag tttaaagtag ctttttccaa ttctgtgaag aaagtcagtg 24120 gtagcttgat ggggataaca ttgaatgtat aaattacttt gggcagtatg gccattttca 24180 ctacattgat tcttcctacc catgagcatg gaatgttttt ccatttgttt gtgtcctctc 24240 tgattttctt gagcagtggt ttgtagttct ccttgaaaag gtccctaaaa cttcatttct 24300 aaaaaaaaaa aaaaaaaagg aatatgctac caaataggat tctttaaatc aattgtctcc 24360 tccttcagaa aacatccaga acctgacctc ttatcaccac ctccactgac cactttggtt 24420 taagccactc ttattcccct tcggatgtat tgcagtggtc tcctgacagt ctcatttcta 24480 tccttacctt cctagattcc agaggttgag agtctgccct cgacttagaa gccattgtga 24540 tactgttaaa atataagtca gatcatgtta gccgtctgct ccagtgtctc cagtggtacc 24600 atatctttta gagaaaaatc aaatttctta cagtggcctt caaggcccta cagcatctgc 24660 actctgctgc actctgattg tactatctac tactcttctc cctggcttag ccagctctag 24720 ccttactcac atcctcaaac aatccctgtc atctcttagc atcttagcaa tttcttcttt 24780 cctctctaat tcctttcccc aacactcccc acttctccaa cccattccag cttccagctt 24840 ccagcttcca tgagtatcct gaaacataac aaactttggt tactgccagt gtctcactta 24900 acacattccc ccactacagt gttgtcaagt tgtttcattt aacaccagta acatttaata 24960 tcagtaatag ccagattcta tcttaggaaa aatatgccta aaatttattt gctgaaaaaa 25020 aaagaaaaga aaaatttgga taataatctg ctttgagtta ttacaaacac agcagtatct 25080 gagaagcaag actgagtgtc ataaggaaac aaccagatta aataaaagca tagctggatg 25140 agataagaaa atactgcagt gtgatccaaa atgcagtaac agagttaaaa tccatattgg 25200 tcgtcataaa gagcaggaaa taacaccaat tatgtagagt tcacatttaa ggagttactt 25260 cggaatgaag agaaattggt aaactggtga aaattacaca ctatgattta ttgtttttat 25320 tatgtaccag tcattgtggt aacatttcat ctaatttagc tcacttaatc catataacag 25380 ccttataaaa tgtttatctc tatattatat gtgggaaaaa ctaagatttt gattaaacaa 25440 ttttatcatg ctgcagaaag tgacagaact gaatttgaat gaaagtctat ttgactctaa 25500 agcatatggc cttatcacta tgctgttgga caacagatac gatgaataga gagcacatat 25560 gcaacccata aatatccagt gattctgaag aagaaactaa aaaacaaggc agcaaaagta 25620 ttaattgatt atagaaaatg actcttcctt atgtatcttt tttacgagat catttctaca 25680 ttgtgtcacc agcacctaga acaatgttgg cacatagtag atattcaaga aatatttgtt 25740 tattgaactg tcataattac tgttaagcat ttattctgtg tctgttatgg tgctatagat 25800 tttatataca ttattttacc ctcaaagcaa cttttaaact gttattttat agatgaggat 25860 aatgtagtgc caagaagtta aatgatctgc tcaaggttat atagctaata ataggtggta 25920 gaacacgtat aacagaagta agtttagcag ttaaataaaa attttacttt ttagatcaaa 25980 aggactcact gcatcccagg taaatgtaat tttaaaaagc taaaactttg caatattggt 26040 aattttttaa aatttttcat gagaaggaaa aacaccctat atgcatttaa gtaggattaa 26100 taaaactggt taactactaa gacataaaaa ttgggttgac attagatttc ctctttgagt 26160 actaaatgct gagattggca aaaattccat caggttgagt tgctattctt gggtgaagat 26220 aacatatatt cttatatatg tgagaggtca ggaatatgcc cagtctttac cgaagtactt 26280 gtttgtctga tggttggact aatttgggat tcctgtagtg tcctgtagtt cagggtgaag 26340 tttagtaaca tgcgggggta agagacagga ggtgctattc tctgccctgc tgctcaccag 26400 tgtgctgtat ccctcctcag gatcaccctt aaggtctcag atgctcacac ctggtgttca 26460 gtgcccagtg gtctgttctc tagccagtat attctgtgtc gtcttagatt ccagtccaaa 26520 gactatttag atctgctcca tccttgggaa tttccacgca ctttattatt tcttctaggg 26580 caggggtccc caacctagaa caagcctacg atttgtgggt cataccatac tgactttcac 26640 catttcactc cttgaactat tgaacataca ttatcatttc tcctgccatt ttactcacct 26700 gctgctcacc tcctgctgtg aatcaggcca cacagcagga ggtgagcagc aggtgagtga 26760 gcattactgc ctgagctgca cctcccatca gaccagtggt ggcattagat tctcatagca 26820 gcgcaaaccc tattgcaaac tgcgcgtgcc agggatctag gttgtgctcc ttatgagaat 26880 ctaactaatg cctgataatc tgaagtggaa caatttcatc ctgaaatcac cgcttccccc 26940 aacccccccc acccctcact gcccccagtc cgtggaaaat tcgtctttca tgaaacctgt 27000 ccctggtgca aaaaagatta gggactgctg ttctagagaa attaaatgtt tttatgtcta 27060 ctttgttaca aggttacatg aaataaacca gttaaatgct tagtatattg gtctctaata 27120 tactcaataa gattttcctt ctctccttca ccccacacct ttttccttca cttttatcaa 27180 aaatccttgg atcttcatga agatatttta aaacctactg atttgttatt gtccagctgt 27240 ctttcacact ctgctagata gtcactcttc attccacttt acatgaccat cccctgactt 27300 actaacgtga aaaactggtc tgaacctttg tctcattctg catgcatctt cagaatatgc 27360 tggattccag aaaacataca aaaaacttta aggaatagta cagcgaatat ctgtaatcaa 27420 caatttgaag gtaagttgca gacatcatga catttcacct cttaaggctc caaaattcat 27480 gtcctaagga caagaacatc ttccattaca accacaatat tatcacccca aaattttaac 27540 attgataaaa caattctaat attcagctag tattaaaatt tcttcagttt ttcccaaaat 27600 gttttgtata gaatgttttt ttattcagga tccagtcagg aattgtgtat tttacttggt 27660 tagcaactct gtttagtttt ctttaatcta aaaagatctc ccaccttttt ttgtctttca 27720 ggacattggc atttttttgg gagtctaggc cagccgtttt gtagaaagtt ccataatctg 27780 gctttgtctg atttttcccc cacataatta tatgccagtt aaacgttttg acaagaatcc 27840 tacacaggcc aacggttctc agacactgat ctcatgaccc ccttatgtgc ttaaaaattg 27900 ctacaagccc aaagagcttc tgtatacgtg aattctctct ctcagtattt actgtgttag 27960 aaattaaaac tcataatgtt ttaaaacttt ttattagctc atttaaaaaa cagcaaaccc 28020 attagatact aatttttcaa accaaaaaat ttaatgacaa gaatgacatt gttttacatt 28080 tttataagtc attttaatgt ctatcttaat agaagacagc tggattctca tatctggatc 28140 tacagtcaat ctgttccaat atgttgtttt ggtggaagca tgggaaggaa atgtggcctc 28200 acacagatat gtagttggaa aatggaggag tattttaatt gtcttttcag acttgacaag 28260 tggtggccat ttcattagga attgcaaaag gatgattctc tatttctatt atcccttctg 28320 attttatcag ctataaatat ttttaatagt tctctctctc ttccaagagg caatctggta 28380 atcttaaaat acacttaata atggaaagcc taacagatac ttattattta cttttaattt 28440 tcagttttca aagttgttaa gtttgtgccc tagtaatctc cagtggtttc cagtgagttc 28500 ctcttgtttc tttcattctt tctcttctta catatcatta tgtactcatg ttttttaaaa 28560 tatatattca atgtgtttta attacttgaa ccagttcttt ttgatgttca agtggtttta 28620 attttggttg gtgtgaatta tcagtggtca atgaaacaca attttattag ccctcagtaa 28680 aagcaaatca tctgtcacag caaagcatcc caggctcaac ttgcacattt cctgccctag 28740 tcctggatca gtcatttccc caaggagctc tggttcgttt tagatgaaaa ttatattaca 28800 tgtgaaaatc tgaattcctg ctggagttgt ctttactttt gggcattttc agtgaagaga 28860 cctagaaaat acctcttttt gaaaaagtaa ggggatcatg agtttatacc atgatttctc 28920 aacctcacac tattgaccta ttgggccaca taattgttgc agggggggat gtcctgtgca 28980 ttatgggatg ttattagcct ttttggcctc tctgcaataa tacctaccct ctagttgcat 29040 aaaccaaaat tgtctccaga tattgtgaaa tgtcctggtg aggaagggag caaaattgcc 29100 tctagttgag aattactggt ttatgtttgt atttctagca aaatcaaaat aattactagt 29160 tgttttatct gtatgtattg tgtgtgtgtg tgtgtgtgtg tgtgtgtgga ttcaaaatac 29220 tagtatcagc attattacct ctaacaacaa aacctgcttt gtagactagt tcttccatct 29280 cctgcataaa atcttaggcc agtcccaaag attttaatca gaagaacaat ctcattccac 29340 taaccgtatt agtctgttct catgctgctg tgaagaaata cctaagactg ggtaatttat 29400 aaaagaaaga ggtttaattg actcacagtt ccacatggct gggaaggcct caggaaactt 29460 acaatcatgg cccaagggga agtaaacatg tccttcttta caaggcagca ggagagagaa 29520 tgagtgccag taggggaaat gctagactct tataaaacca tcagatctca tgagaattca 29580 ctgattgatc acactgctca tgatgatcac gagaagagca tggggggaaa ccacccccat 29640 gattcaatta cctcccactg agttcctccc acaacatggg gggattatgg gattacaatt 29700 caagatgagg tttgggtggt gacacaaagc caaaccatat cagtaatcaa aatgcccagg 29760 gaagcattat gccaaattta caaatcctct tctaagttta gatttcttct ttgccccaat 29820 tcagcactca ctgctttgag cttgaagcct aaagggaaga acaaataaga tctgcttctt 29880 ctctgtttat gctgcctaca ctcacccaat gacagctgat tttgaccctt ccagggccaa 29940 cgtgtgggaa gagaagttag agagcacagt tttacacaac aagtaggaat tttagtgtca 30000 ctggggttat ctgtgtttgg tgggtgatta caggctgctc ttcctctagt gggatacttt 30060 ggtgagattc atggagatac tttactgaga gcctctaact gtaatcctgt aatgctgtaa 30120 tgcagtagac acctctcttc tctctggaca tgaccgtgac cctctgcttc tggacttccg 30180 ctgactgacc ctacacagat tctgttggat cacaatactc tctaggttgt cctcttagct 30240 ggagccagtt tttaaaagaa agaaactttt attgataaat attttcagaa aagcactcaa 30300 agatgtagag cttatcagta ggtaaatgaa acgatgttat tatcacccag accaaaaaat 30360 ttaaagatca ttaggttccc agaagctcca ttttgcccct tccaatcatt ccccaaaagt 30420 aaccgccatc ccaatttcta acatgataga ttggtttgaa ctattttaga actttatatt 30480 aatggaatca gatagtattt ttctatgtct gcttatttta cacaacatta tacttatgta 30540 gttcatccac attttgcatg gagctagaca tttattctca ttgctgtata ataatgcatt 30600 gaataaataa aaccatttgt ttacacatta tgctgtaggt gatcatttgg gttgttttca 30660 gattgaggct attatgaaca ctgctgctat aaatattctt gtacaaatct tttatttttt 30720 gcatgtggac acacctgtcc ctacagataa gcttttcatt tgcctctcca aggaccctgg 30780 caatttcact ggtcctgtac cagtctttgt tccttggccc aggttcctat accatgtgat 30840 agtgaaaatt cacctcccat taaaggccaa tattttattt tgtttatgtt gtttttggca 30900 taaatgattt ttgtcctcac cctgaatttt ggacagattc ctggctgcct ggcattttct 30960 gtgtagtgtt ttctagccct gttttcattg ctaaggcagt cttttaaggt tctacatttt 31020 tatgcatata gttcagccct gccactctct tccattggga ctgaagtaat atatcccatc 31080 ctctcatggg ctttaaaccc catttcttat cttttaggca tttcttatat ctgtatctga 31140 aacttttttg gatcactgca gagtccagtt aagtttgttc ctccagcttt catttctttt 31200 ttcttttctg gcttatagag atttacctac ctttttttcc agactgaaat taagcaacat 31260 gcttctatta tattttatcc agtatttctg tgtttgtggt agaaagattg ccctcatatg 31320 gaccagctca tgttgcttca agcccgtgac ttgtgggtca taccatactg actttcacta 31380 tttcattcct taaactactg aacatatatt atttcctgcc atttgactat gtattttccc 31440 cttcttttca aaaatgccat gaaaaattgt gaactctctc agacctaagt gaactatgtt 31500 cacttgtgct catattgaga agcaacacaa acaatatagg tctcttattt tacaggtaag 31560 tccctttctt gtttctaata aagatatttt atttcctaat aaagatattt tatttccttt 31620 aaagtggaat tttgtcatct aaaatttttg taagtcacta ggcaaataaa atgatctaat 31680 cacattttga tagtgctttt attttcacag agtatttttt attaagagta acacttgggg 31740 ctgggcacgg tggctcacgc ctgtaatctc agcacttcag gaggccgagg caggaggatc 31800 acaaggtcag gagatcgaga ccatcctggc taacacggtg aaaccccatc tctactaaaa 31860 atacaaaaaa ttagccagac gtggtggtgg gcgcctgtag ttccagctac tcgggaggct 31920 gaggcagggg aatggcatga atccaagagg cagagcttgc agtgagctga gatggctcca 31980 ccgcactcca gcctgagcga cggagcgaga ctccgctcaa aaaaaaaaaa aaaaagagta 32040 acacttggaa ataatcttgt tagacttcac atttaattat ggtctattac tataaaaagg 32100 tgagaaatta ctgaataaaa agcattctgt aagaaaaaaa gttaaaaggt ggtaaaaaat 32160 cagaagcaag tgactattta ctatatatga caataagtga catatgtctt atactgttga 32220 gtataactat atttacaaat ataagcgaaa caataggtat aattgtatat acacctatct 32280 aaaaaatatt tatcaagctg tattttacac aagagttaaa agtatcaaac tgagtagttc 32340 ctactcttat agaacctagt agctatagac acaaagtata caataggaat acattgaatg 32400 tgcagttggt acacagaaga aatgtctaaa gcctttatga gagcagtcag gtaagaccag 32460 atgaggggag cagttaagcc aaggcttgaa agacgagtga gattttgtta agttgataaa 32520 ttggaaggaa gggaggaaag tcatagaggc atgagagagt ttgggggtaa tccaaggatg 32580 ttagtactct ggagcataaa gaacatggga aagagtatgt gattagaaag aaaagacata 32640 acagtttgaa ctcaccccct gctttcccca ccctaaaata gatcagtttt gcaaggagaa 32700 agaagcatag tgctctatta agctccccag gtttcaacag gggcagatga tgcatatatt 32760 aagggagaga gctagatagt ggctcctagt ggggtgaact cccctattta tcattatgcc 32820 atgtaacctg tttgtggtgg cttatagctt ataagtgttc ctaaatgtct tataactcct 32880 ggtggtgata atgtaggtaa acagcaaaga tggcagcaaa tggaatgctc tgagtggttt 32940 aatatagtga cgtgttttac acttgttgta gaatcacaat gttatgttgg actgaggaga 33000 agaaataata cgcaatttga gccatcaacc tacggtttat gagtgagtga gtggatctag 33060 acagggatgt gtttctcaaa accagaatat aaactgtggg ctgttgtctg aagaaaatgg 33120 tagcaactga tgttatgtta gattttcatc tttttctaga tttccaggaa aatgttgtga 33180 cagggctaat gcacttgtgc ctgacctttc ttcacctgca tatacagcca ggtacctgaa 33240 gtagcgagtg gctcccactg cattcattgc attcttggtg gctctgagtc ctggccatgc 33300 attatggttc agaggcaatg cctgggcccc atgccaaatt cccttagtcg gctctagttg 33360 gaactggctg gctacaacat ttctttaagc caaaacattg cttgagaaga taaaccttta 33420 actgtactcc ccatattttg tttgcatgat aatgcatcta ctttgcagtt ttgatggctc 33480 atcacttaat gttaaagatg atgaaaagtt agagtgaatt gagcttccag atttctttaa 33540 gccattccag cctgtcacag gcagtctaca atgatatgag tcgtgccaac ccctcaaggt 33600 cgtgacttca tgccagtgcc cttaattatt aaaaattatc cttccaactc aaactttaaa 33660 tattggacat ctaagtcatt ttttcttcca aacttataag aattgcattc tgtttggtaa 33720 ctataatttc cttatttata ctcatgttta aatttatact tatgtttaaa tttatttggt 33780 acttatcatc agtctcttca taccagactt cctacctcat acattgtctg ctataacgta 33840 tctgttgttg actggattac atcttttgga agagcatact ggggtcttat ttcccaattt 33900 catgcaagtt tgagaatatc tttaagttgc ctttaaaact gaataacttg actagatata 33960 gacttcttgg accacatcta gaaaaaatct aactatattt gagatttaga tgtgtgataa 34020 tcataattat aatagacatt tgttaagtac ttgctatgtg ccagactatt ctaagctctt 34080 acatgagctt gtgtattcct tagatccaat ccaaggtact aatgttatct tcattttaat 34140 tgccaaatgt catctagcta ctaagtgatg agattgacat tcacacttaa acagtaactt 34200 tctatactac ctctcaagat aattaaccta aaatgtttta actgtgtgac cttgagcatg 34260 ttatttaact tctagatgcc tcagttaact ctgtataaaa tgagaatatt aatacctcca 34320 aaggttgctt tgatgatgaa atgagttaat atatgtcaat agctcataac aatgcctggc 34380 acatagtaag tgcttaataa ttgtctgttg ttcttttatt atttttattt gtggatagta 34440 agttgcagat ctgagctttg aacaagcagt ctggcctctg taactttgct cttcggtact 34500 acaatgtagt gtcagcaatt aagatggcat tttaatttag tgggggaaaa catggattag 34560 tcagtaaata gtgttgggat atagtagata ctcaatactt gttgaatgaa tgagagtttt 34620 ataaaagtat aaatacatta acttaaatat taacaaatag atattaaata ccatttagat 34680 ttgtatagac agtttggaaa tactttagtc tgttccactg caataaaaaa ataccttaga 34740 ctgggtagtt tataaataca gaaatatttt ctgatggttc taaaggctga gaagtccaag 34800 atcaaggtgg cacagattcc atgtctggta aaagcttgct gtcttcttcc aagatggcgc 34860 cccttgctgc atcctcacat ggcagaaagg ataaaaagga ccaaattcac tccctcaagc 34920 ccttttaaaa gggtactgat tccatccatg gaggcagaga ccccacctct taatattgtc 34980 acattgggga ttaagtttca atatgaattt tgaaggggac acaaacattc aaaccatagc 35040 aagaaggaag caaaaatgag aatagatgca tgattatcaa attgtaggtt attttctgtt 35100 ttgaatttcc ttacgttctt atataatttc tctaataata caattcatga tataatttgt 35160 acagataacc acttagttgt attttgtata tattgagttt gaggtaccta gaaatatgac 35220 tggaaggtca gagctcatat tgagattttg ggaccatcag aagataatga gacagactac 35280 ccacaaagtg tgtgcagtgg cacaccacat ataaggaata agcaaagtaa gaggagttga 35340 caaagggaat ttaaaaggaa gtgctggaaa gacaggtcaa gagtcagaaa agagaggccc 35400 caaaatatga atcaatggga caggtgggga aagaaattac ctgaagcaaa tgattcaggt 35460 caggaaagca ttcacagcat ttaggaatga aaaaatattt gatgatgtta agccatttta 35520 taagaatagt gaatatacaa gtcaaattac cacataggaa taaaggaagt taggtagaaa 35580 cagcaaatgt agatgatgtt ttcgacaact ttgaggacca aaaaaagggg aatgtattca 35640 gggagaatct agagtgagct agggttcctt tttttggtgc tggaactttt ttggatggaa 35700 aaataatgag agagcaggac atcactggac tggagagact aaagacttta ggagagaaag 35760 gaggaaattg atggagcaag acttgaaagc agataggaag gaattgtttg aaggacaaaa 35820 gcaaaaacca ttggtaagga ggactctttg agacactgaa taaggagatg atatcgtagg 35880 ccattttaga aatgtaagtg agacagatca gcagcatgcc agcttctggt gtttagatca 35940 gacctttttc ctgcgtattt atgaccaagg agcagacagt aggacagcct ctcttcaaac 36000 aattacatct gcgtgctcta tttctcgaag tctgttttct gtatagagaa aaccttggtg 36060 ggcgtggtgg ctcatgcctg taatcccagc actttgggag gccaaggcgg gcagatcacc 36120 tgaggttggg agttcgagac cagcctgacc aacatgcaga aaccctgtct ctactaaaaa 36180 tacaaaatta cccaggcgtg gtggtgcatg cctgtaattc cagctactca gaggctgagg 36240 caggaaagtt ccttgaaccc aggaggtgga cgttgtggtg agccgagact gtgcctttgc 36300 tttccaggct gggcaacaag agcgaaactc cgtctgaaag aaaaagaata gaaaatcttg 36360 ctattttctg ttctctcttc ttgtgagata tcagtatagg gtagaaatag atgatcaact 36420 gatagactga cttacacaca ccttgctctc acagtcactc ctatagccta aatatataac 36480 ttgtggtaaa aaaaaaaaaa attagagtca aatcagaaag tagtatgcag tacactctgc 36540 taacctcctg taggactggc actatgtgca caaaagtcag caaagatatg aggcttcaca 36600 gttacccttt attaggtgaa ggcttaattc ctgggactct aaacaagaga tgaacactat 36660 gtgagtgtac atgaacataa gtttatagag gcatgcatct ttaagaaaat atttgtgctt 36720 ttccagaaat gcatgaggtc aggaaccttc ttgatatcag ctgtcacata tataggcctt 36780 ttggcatttt tgccctttgc cgtttaggct aatcctttcc cctgcttgat taagtacatg 36840 ttttaggtac ctctgagttc ctttactacc aactttggcc agaagactct gattttaatc 36900 ttaactaatg taaaatacta caggaaaatc ccatgatctt ttaaaattat gtgtcatgct 36960 tcagcagtat aagtaggctg aagtaggctg atgatattaa aagactcaca gtctgtttta 37020 aagaaattta ttgggaaaaa aagagagtgt caaaaatagt aaattaacgt ttacatgttt 37080 aagctgaatt tttttatttt tgttaaatct aaataaatta gattgttccg tgccttatta 37140 acatatttgt ggtttaaaaa tcattattga atattaaatt catgtgccag acctttttaa 37200 aggcatccat agtttcctct acacaaagtg accccagggt gatgtgcgat gtgtattgtc 37260 tatacttcta taaagtttct cctctttatt gagtacttct atctgtttta agttcttcct 37320 ttatcattat ttctgttcaa ggtattttct aatgtgtagt caaaataagg caggtattaa 37380 attcagtaca ataagtgatc ttttttacat gctcttccat tgattcctta aagagagaac 37440 cagctctggg atttttccaa tagaactaag catttagatt tttttttaaa aggtcttttc 37500 cagaggcctc ttcaattctt tagtctgtat ctgttttctt gaaggcatca atgtcagaaa 37560 gaaaggtttt actagagttt gaccacctgg tacaactaat gcttatcaga aacgtgtagc 37620 taatcttgaa atgacatcta aaggcaatct gatactgaca ccattaagaa gataaacaga 37680 aatctcttgc aaaagatttt taaaggagcc ctaggattga ttcttcagct atgctcacct 37740 aatttaggct cctaaaggca caggtaaagt actgagcagt tttatagctt caccaacatt 37800 cagctgtgcc aggcaggaag ctggctgagt catgcacttg ctgatgtgta tctagtctct 37860 atttcagttg tcactgcata gaatttccag tgaatcttag atattgtaaa taattttgac 37920 tcattttgtt tttttagggc agcacaacta cttatgtgct ggaagaaatg actgcatcgt 37980 tgataaaatc cgcagaaaaa actgcccagc atgtcgcctt agaaagtgct gtcaggctgg 38040 catggtcctt ggaggtaatt ctgatgtttt catcaataat atactgtgta atctttatac 38100 tataaaactg tgtgtcagaa aattgcaatt tcttattcat ctttgttttt gttttttctg 38160 tcttgattat tggcagtgac tctgcacatg tgagtgtctg aatgaagcaa ccaataccat 38220 tccattaact ttacagtttt ctagcacatt gaggtctgtt aagagaagaa aactcaattc 38280 atctatattg cctataagta gaggtaatgg gcaatgtact gacctgtcat taagtaacat 38340 gtgtgggagt ctaggctttg ctgcccatct cctgtgtgac cacagctctt cctgggcatc 38400 agtctactta tctgtaaaac gagagcatta acattccttt cagctcaaaa aattgactct 38460 gcaaattggt caggtacatt gcttactagc ttctccaagt gattgattaa agcaagatta 38520 ctgtaactgg ggtaggaatc catttgatga agagttatat taataattcc cttttcccat 38580 atttttatta gaatctctaa aactttcagt aatatagcac acatttaaaa aattaatgtc 38640 agatagtgac agatagtttg aatgaaaaca tagcagagtg agggagatca agcgagccac 38700 agttatgcaa gacacccgta ctcccttgaa ttttttcact tagccacatc ctgcacaaca 38760 ctgtctggga atcgtagctt ttacatctag atataacttt ttaaaaacat aataaaaata 38820 ttacataatt gttaaagcta aaaatgtttt ttcagtgcac taactataaa cagcttgtgt 38880 tccatggacg ttgtgtgtat tttcctttgg gaaatgctat atttagacca atgcttgtca 38940 agtttaaagc gtatacaact gtcacctcat ttgtttttgc aaatgtgctg gaccaaaatt 39000 aatcacccag gtaacatact gggcacttta cagtaattca tgtaattaga tcacttatag 39060 aattttataa tttcttgatt aaagatttgt tttcctaagt taagggcagt gattaggttg 39120 acagtagtgc ctggcttaag gaggtgctca gtaactttct attaaatgaa taaaaccaca 39180 acctcagcag attaaaattg tttttgagtc ccacacctca attaccacct ctttgtattt 39240 ctgtttcaac tcagaagtct taaggaacct cctaaatttt agaaaataca actttttcac 39300 aaaatcacac atctagtcaa tgtcggagcc agaagaaaga cctgaaggta ttctgaccct 39360 ggcgcagcca ttgttttctc cttggcatgc ttgccttaga aagtagcaga gcatctaatg 39420 ctgacactta gattccatta agatctttaa tcttgagtgt tttcccaatg actttaaaag 39480 actcttcaat tttatcaaag aatgaaaatg gcggagttct ggatagaacc ttcagaatac 39540 tcctggggat cagcaatata acctagagaa atttatgtta tcttttaata ttctagggct 39600 ttatttattt atttttgaga cagagtctca ctctgtcgcc caggctggag tgcaatggtg 39660 caatcttggc tcactgcaac ctctgcctcc caggttccag cgattctcct gccacaacct 39720 cccaagtagc tggtatcata ggcacccgcc attacgccca gctaattttt gtattttagt 39780 agagatgggg tttcaccatg ttggccaggc tggttttgaa ctcctgacct caggtgatct 39840 gcccacctcg gccttctaaa gtgctgggat tacaggcgtg agccactgca cccggccttt 39900 agcatattat tcaagcatct gtaatccttt atcttctcta accaaactgc ttgttacttt 39960 agcctgttat tcctatgctt cccaagccat attctacggg cttcacagct attgctgcat 40020 tttcatacct tttatattac ttatttgttt aactctactc cctccatctc tccctccttt 40080 ccgtccctcc ctccctccct tccttcttct ttccttcctc cctcttctcc ctcaatcgct 40140 ttctaatttg caccccatcc ctttctttca gtaactgtat atcgagccct ttctagttac 40200 cagatactat tctaagttct gcgcatatag cagcaaagaa aatggacaaa attcctgccc 40260 tcatagatct tacactgtgg gaggacgagc aacaaatgta catgtcaaat aatgataagt 40320 gctgtggaaa gatattaagc agagttagtg gggattatca ggaagcagac tcactactaa 40380 actcattagt ctaatatttt tgttttgctt agcctcttcc taagcaataa tatctctgaa 40440 ataatggatt tgatatgcca gttataattt ttctaaaaga cattaatata cattcttaac 40500 tatttaaata tatattcgat gtaccaccta aaaactgctt gtataccata aatgattatg 40560 tgttaaactc tgggaaagag tgtattagac catttttttc cagctatcgc agctctattt 40620 attttttcag cttttatttt agcttcagtg ggtacatgtg caggtttgtt acctgggtat 40680 attgtgtgac actaatgttt gaggtacaaa tgatcccatc acctaggtac tgaggtactg 40740 agcatagtac tcaatagttt ttcaaccctt tcttcttctt ccttcctcct ctagtagtcc 40800 tcagtttcca ttgttatgct catgaatacc caatgtctag ctcccactta taagtgagaa 40860 catgcagtat ttggtttttt gttcctgtgt taatttgctt agggtcttgg cctacagcta 40920 catccatgct gctgcaaagg atgtgacttc gttcttttca tggctgcata atattccatg 40980 gtgtatatat gccacatttt ctttgtccaa tccactatta atgggtgcct cagttaattc 41040 catgtctttg cttttgtgag tagtgctgtg atgaacatgt gagtgcatgt gtctttctag 41100 tagaacaatt tattttcttt tggctatata tccagtaatg ggattgctgg tctaatggta 41160 gttctgtttt aagttctttg agggccggga gcggtggctc atgcctgtaa tcccagcact 41220 ttgggaggcc aacgcaggtg gatcatgagt tcaggagatg gagaccatcc tggctaacac 41280 agtgaaaccc cgtctctact aaaaacacaa aaaaattagc caggcatggt ggcaggcgcc 41340 tgtagtccca cctacttggg aggctgaggc aggagaatgg catgaacctg ggaggcggag 41400 cttgcagtga gctgagttcg cgccactgca ctccagcctg ggcgacagag cgagactgtc 41460 tcaaaagaaa aaaaaatgtc ttttgagaag tgttcaaacc aatttctacc gtggcagaac 41520 taatttacat tcccaccaac agtatataag tattcccttt tttccacagc ctcaccagca 41580 tctattaggt tttgagtttt ttgagttttc ttttttgaga cagagtctca ctctgtcacc 41640 cagactggag tgctgtggca caattatggc tcactgcagc ctcaatctcc tgggctcaag 41700 tgatcttcct acctcagcat cctgagtagc tgagactaca ggcacgcacc accacacctg 41760 gcaaactttt tatttatttt ttttttgtaa cgataaggtc tcactttgtt acccagtcag 41820 gtctcaaact tctgggctca agtgatcctc ccaaagtgct ggaattacag gtgtgagcca 41880 ccatgcctgg catgagtttt taataatagt cattctgatt ggtgtaagat ggtatgtcat 41940 tgtcgctttg atttgcatat cttaatgatt agtgatatgg agcattttaa atgtttgttg 42000 gctgcttgta tgtctccttt tgagaaatgt ctgttcaagt cttttgccca tttttttggt 42060 gagatttttt ttttttgctt gttcaactgt ttaagttcct tatagattct ggatattaga 42120 catttgttgg aggcatagtt tgtgaacatg ttctcctatt ctgttggttg tctgttaact 42180 ccattggtag tttattttgc tgtgcagaaa ctctttagtt gaattaggtc tcacttgtta 42240 atttttgttt ttgtttcaat tgcttttgag aacttagtta gaaattattt cccaaggctg 42300 atatccagaa tcatatttcc tagattttct tttaggattc ctatggtttg aggtgttata 42360 tttaaatctt taatctacct tgaggtaatt tttgtatatg gtgaaatgta ggggtccaat 42420 ttcattattc tgcatgtgac taggtaggta tcccagcacc atttattgaa taaggaatcc 42480 tttctgcatt gcttattttt gtcaacattg tcaaagatca gatggctgta ggtgtgcacc 42540 tttatttctg ggttctatat tctgttccat tggtctatgt gtctgctctc gtgccagtat 42600 catgctgttt tggttactgt agccttacag tatagtttgg aattgggtaa tgtgatgcct 42660 ctgacttttc tttttgttta ggactgctgt agttatttgg gctcttttga ttttatataa 42720 attttagaat agctttttct agttctggaa aaaatgtcgt tggtagtttg ataggaataa 42780 cattgaatct gtaaattgct ttggacagta tggccatttt aacaatactg attcttctaa 42840 tccatgaaca tggaatgttt ttacatgtgt tagtgtcatc tgtgatttct tttagcaatg 42900 ttatgtagtt atccatgtag aaatctttca tctgcttagt tatatgtata cctaggtatt 42960 ttgtgtgtgt gtgtggctat tttaaatggg attactttct tgatttggct ctcaccttga 43020 atgctatagt tgtatagaaa tggtactaat ttttacatgt tcattttgtg tcctgaaact 43080 ttactgaagt catttatcag ttttaggagc cttttggtgg agtctttagg gttttctagt 43140 tataaaatca tgtaatatgc aagagagata gtttgacttc tttttttcat gtttgaatgc 43200 cttttatttc cttctcttgc ctgactgctc tggctagcac ttccagtgtt aacaggagtg 43260 gtgagagtga gcatccttaa ctttttccag atctcaagga aaattcttcc agcttttgcc 43320 cattcagtct gatattggtt gtgagtttgt cattttggtc tcttattgtt tttaggtata 43380 tttctttgtt gcctagtttc ttgagggttt ttatcatgaa gtggtattgc attttatcga 43440 aagctgtttc tgtgtctatt gagatgatca tatggtttta tttttaattc ttctcatgtg 43500 gtaaatcata tttattgatt tgcatatatt gagctaacct tccataccag gagtgaagcc 43560 aacttgatcg tggtaaatta actttgatgt ttgatttggt ttgctagtgt tttgttgaga 43620 atttttgtgt ctgtgtttat cagagatatt gtcctatagt tttcctttct catggtatct 43680 cttacaggtt ttggtatcag ggtgatactg gcttcagaaa acgagttagg gaggagtctt 43740 tccttctcga ttttttgaaa tagtttcagt agaattggta ccagctctac tttgtgcatc 43800 tggtagaatt tagctgtgaa tctctctggc cagggctttc tttttggttg gtaagttttt 43860 tattactaat tccatttgga acccaatatt ggtctgttca gtgctttagt tttttcctga 43920 tttaaacttg ggacattgtg tgttttcagg aatttattca tttcctctag atattgtagt 43980 ttgtgtgtgt agaggtgttc acaatagtta tctaaggatc tttcatattt ctgtgggatc 44040 agttgtgatg tcacctttgt catttctgtt tgtgtttatt tggatcttct ctcttttttt 44100 ctttgttaat gtagctagca gtctatcaat cttatttatc ctttaaaaaa ataacttttg 44160 gttttgttga ttctttgtat agatttttgg gtctcaattt catttggttc tgctctttag 44220 ttcgtccttt tcttctgcta gctttaggga tagtttgttc ttgttttttt tttttttttt 44280 ttttttttag ttcctctagg tgtgatgtta ggtcattaat ttgagatctt tctaactttt 44340 tgaggtaggc atttagagct gtaaactctc ctcttaatat tgtttttgct acattccaga 44400 gattttggta tgttgtgtct gttttcattt attttaattt ttttttattt ctgccttgat 44460 tttattgttt actcaaagtt atccagagca aactttttaa tttccatgta attgtgtggt 44520 ttgtacagat cttcttgatg ttggtttctc tttgtattcc actgtgacct gacagtatga 44580 ctggcatgat tttgactttt taaaatgtac tgagtcttgg tttatggcca aggatgtggt 44640 gaatcttgga gtatatgttc tgtgtacaga tgagaagaat ttacgttctg tggttgatgg 44700 gtggattatt ctgtagatgt ttattagatc caattggaca agcattgagt ttaagtccag 44760 aatttctttg ttagtcttct gccttgatga tctaatgcta tcagtccccc actattgttg 44820 tgtggctttc taggtcctta gaagtccttg ttttatgaat ctgggtgtgc cagtgttgag 44880 ggcgtatgta tttaggatag ttaaatcttc ttattgaact ctttatcgtt atgcccttat 44940 ttttcctttt ttactgttgt taaagtctgt tttatctaat ctaagaatag caacccctgc 45000 tcttttttgt tttctgtttg catgctacat ctttctccaa ccctttactt tgagcctatg 45060 gttgtcatta catgtgagat gggtttcttg aatacagaag atggatgagt ctggttttcc 45120 tagctggttt gccactctgt gacttttttt tttttttaga tggagtctta ctctgatgcc 45180 caggctggag tgcagtggcg caatctcagc tcactgcaac ctccacctcc caggttcaag 45240 caattctcct gtctcagcct ccggagtagc tgggattata tgcacctgcc accacggctg 45300 gctaattttt gtatttttag tagagatggg gttttacctt gttggtcagg ctggtcttga 45360 actcctggtc tcaggtgatc cacccgcctc agcctcccaa agttctggga ttacaggtgt 45420 gagccactgt gcctggaccc actctgtgac ttttaaatgg gacattgtat tagtccattc 45480 ttgtgctgct ataaagtact acctgagact gggtaattta tgaagaaatg acatttaaat 45540 gactcatagt tctacaagct taacaggaag catagctagg caacgtcagg aaacttacaa 45600 tcaaggcaga aggtgaaggg gaagcaagaa tatcttacca tggcagagta ggagaggtgg 45660 gtcaggggag tgccacacac ttttaaatca ttatatcttg ggagaaccca ctcactatca 45720 tgacaacagc atgggggaaa tttgcctcca tgatccaata acctcctacc aggttcctcc 45780 ctcaacattg ggaattacaa ttcaacgtga gatttggatg gggatacaga gccaaaccat 45840 atcattctgc ccctggcccc tcccaaagct tatgtccttc tcagttttca aaacacaatc 45900 atgccttccc aacagtcccc caaagtctta actcattcca gcattaaccc aaaagtccaa 45960 gaccaaagtc tcgtctgaga caaggcaagt cccttccacc tatgatcttg taatatcaaa 46020 aacaagttag ttacttccaa gatacaatag gggtatgggc attgggtaaa tactcccatt 46080 ttaaatggga gtaactggcc aaaacaaagg agatacaggc cccaagcaag ttcaaaaccc 46140 agcagggcag tcattaaata ttaaagctcc aaaataatct cttttgtctc catgcctcac 46200 atccagagca tactgatgca agggatgggc tcccaaggcc ttcagcagct ccaccctgtg 46260 gctctacagg atacagcccc cacagctgct tttacaggct ggcattgaat gcctgcaggt 46320 attccaggca cacaatgcaa gctgttgatg gatctacgat tctgaggtct ggttgatggt 46380 ggtcctcttc tcacagctcc actaggcagt gccccagtgg gaactctgtg caggggctcc 46440 aaccccacat ttccctttca cactgtccta gtagaggttc tccatgaggt ctctgcgctt 46500 gaagcagact tctgcctgga catccaggca tttccataca tcctctgaaa tctaggtgga 46560 ggtttccaaa cctcaactct tgccttcttc atacccgcag gcccaatacc atgtggaagc 46620 caccagggct ttgggtttgc atcctctgaa gccctggccc aagctgtacc ttggcccctt 46680 ttagcaacag ctggagctgg agcagctgga atgcagggca ccatgtccca aggctgcaca 46740 gagtagctag gccctgggcc tggccctcaa aaccattaat ccctcttagg cttctgtgcc 46800 tgtgatggga ggggctgctg tgaaggtctc tgaaatgccc tggagacatt ttccccattg 46860 tcttgactgt taacatttga ctcctcttta ctaatgcaaa tttctgcagc aggcttgaat 46920 tgctccccag gaaatgtttt tttgtctctc tcttttctac cacatggcca ggctacaaat 46980 attccaatat attttttgct ttgcttcact tttaaatgta agttccaggt tcagataatc 47040 tcttttttca tgcatatgag catacatgtt tagaaacagc caggtcacat acatctggaa 47100 tgctttgttg cttagaaatt ttttccacca gataccccaa atcatctctc tcaagttcaa 47160 agcttcacag atctctagga caggggcaaa atgccaccag tctttttgct aaagcatagc 47220 aagagtgacc tttactccag ttcccaataa gttcctcact ccatctgaga ccacctaagc 47280 ctagactcat tgtccatgtc actatcagca ttttggtcac aaccattcaa caagtctcca 47340 ggaagttcca aacattccca tatctttctg tcttctgagc cctgcaaact gttccaacct 47400 ctgcccatta ctcagttcca aagtcactcc cacattttca ggtatcttct taactgtgct 47460 ccactctccc agtaccaatt ttctgtatta gctcattctc atactgttat aaagaactac 47520 ctgagactgg gtaatttatg aagaaaagaa ctttaactga ctcacagttc tgcaggctta 47580 acaggaagca tagctaggag gtctcaggaa acttacaatc atggtggaag gcaaatggga 47640 agcaagcacg tcttaccagg gcagagcagc agagagagag agagagagtg agaggggaag 47700 tgccaccact tttaaaccat catatctcct gagagctcac tcattatcac aacaacagta 47760 tgggggaaat gtgcccccat gatccaatca cctcccatca ggtccctccc caaacattaa 47820 gaattaaaat tcaacgtgag atttgggtga ggacacagaa ccaaaccata tcaggcattt 47880 agaccattta cgttcaaggt taatttttat atgtgaggtt ttgatccctt gtttagttgt 47940 tagctgtttg ctttgttgtt tctattgtgt ttttgctttg tagggtgtgc aggctatgtg 48000 cttaagtgtg tttatgtgga agcaggtatg gttcttttgc ttccatgttt agaactccca 48060 tgtaaggatc tcttgtaagg atggtctagt ggtaacaaat tcctttagca cttgattgcc 48120 tggaaaagat tttatttgtc ctgcacttat gaagcatagt ttggcaggat gtgaaattct 48180 tggttgaaat ttcttttaag aacgctgaaa acaggccccc aatctctcct agcttgcaaa 48240 cctctgctga gaaatctgtt attatcctga tagagttccc tttttatgtg atctgcactt 48300 ttctctggct gcctttaaga ttttttctgt aatattgcct ttgaacattc tggtgactat 48360 ataccttggt gatttttttt tttttgagat ggagccttac tgtgtcacac aggctggagt 48420 gcagtggtga gatcttggct cactgcaacc tccgccctcc aagttccatg gattctcctg 48480 cctcaacctc ccgagtagct gggattacag gtgcctgcca ccgtgtctgg ctaatttttt 48540 gtatttttag tagagtcagg gtttcaccgt cttggccagg ctggtcttga actcctcacc 48600 tcatgatcca cctgcctcag cctcccaaag tgctgggatt acagacatga gtcaccacgc 48660 ccagccagtg atgttcatgt ttgtggtgtc tcacagatgt tctctggatt tctagctctc 48720 tagcaagatt aggaaagttt tcttgaatta ttccctcaac tatattttcc agtttgtttt 48780 ctttttctcc tactttctga ggaatgccaa taattcatag ggtttttttg ctttatataa 48840 tcttatattt ctcaaaaaca gctcatttaa aataattttt ttctttattt ttatcagatt 48900 gggttagttc acaagaccag ccttcaagct ttaaaatttt ttcttctgct tgatccagtc 48960 tattgataac gaatttcaaa atataattga aggctttaag tgagtttttc aattccagaa 49020 gctactattg atttttttta agatgttcat gtctttctta atttcctaga ttgctttaga 49080 agtttatgtt ggttttcaac cttgtctttg attacaatga acttccttgc aatccatgct 49140 ttgaattatt tatgtgtcat ttctgagttt ccatcttggt tagggaccat tgctggaaag 49200 ctagtgcaat cccttagtga tgtcactgca atcagatttt tcattatgcc agaattcttg 49260 cactggtttc ttctcatctg gagacattgg cacttcaaat ttttgtactt attttcatgt 49320 gggtagtagt ttttcttttt tttttctttc cctataatgg tattattatt attattttaa 49380 taattttttt tgtttccctt ttaccccttt tctagggctt gtgcctctaa agaatgctgg 49440 gtaggatctt ttcactttga ttctacagcc ctatgtgctt ctttcagcag gttttatact 49500 gggctctgca ggtcatccca caggcccata ggcggcacgt ataggtaaga gctggctgtg 49560 accaatgtgg ctggatatat acttgatcct tgtttccgag caaaagctct ccattgtctt 49620 aggcaatagg ctgattctga ttcatagaat gcacagtagt tggaactccc tgctcagccc 49680 caaggagaag ggaaccacaa agggcaagtt tggaccgagc aggtccacct gcgtcccctg 49740 atggcaagca caagcaccag tgccaaggaa gaatccagtg ggtggccacc aagcacccag 49800 agttgtacac agacctgaag cttggaaacc tcctcagctg caaattctct gcatgggagg 49860 catcctaagc tcctgattca ggagagtggg tgctccagat gcctggagat ctgctttggc 49920 atggaataga gaggggcccc ctgcaccaag atctctacac aggaggggta aatgacaatt 49980 atatatagtg ctgcaggaaa catgtgaatg cagataatat cttcaatata ccagttttct 50040 ttcttttggc tatatgccca acagtaggat tgctgcatca cgtggtagtt ctgtttttag 50100 ttttaggaac ctccatactg ttttccatag ttacagtact aatttagatt actaccaaca 50160 gtgtacgaga atttctcttt ctccatatcc tcaccagcat ttattttttg tctttttagt 50220 aacagccatt ttaactggag tgagataata tcttattgtg gttttatttg catttccctg 50280 atgattagtg atgttgagca ttttttcata tatctgttgg catttgtatg tcttctttca 50340 agaaatttct gttcattttg cctattttta aatgggatta cttttttttt ttgctattga 50400 gctgagttcc ttatatattc tgataattaa ctccttgtca gatgaatagt tttcaaatat 50460 tttctctcat tctttgtctc ttcacattgt taactatttc ctttgctgtg cggaagcttt 50520 ttagcttgat gaaattccat ttgtcaattt ttgccttgat tgcctatgcc ttgaggtctt 50580 actacaaaaa tatttaccca aaagtcttaa agcagttccc caaagttttc ttgtagtagt 50640 tttgtagttt cagatcttac acttaagtct ttaatcgatt ttgattttat tttcgtatgt 50700 ggtgagtgat agtgatctag ttctgttttc tgcatatagg tagccagttt tcccagcagc 50760 atttattgaa gagattgccc tttccccaat gtatatccat ggcacctttg tcaaaaatga 50820 gttgactgtt aatgcatggc tttatttctg ggttctctat tctgttccat tggtctttgt 50880 gtctgttttt ataccaatat catgctgtta tggttaagaa ttctcatttc tgacatagag 50940 accatttaaa taacaaaaga taaaacatga gagaatatta gaaagctttt aaaagattta 51000 ataaaatctt acagtagaat aatagaggta ataaatatgt tttgacatgt catgacatag 51060 attttgttag gagcagtttg ttcttatctg cttccgtggc tatgttttga ttataaaaat 51120 tatgttttga gttatttgat ttcttcgttg catatgattc ttgtctttaa tgaatcactt 51180 ccctaccttg aacaggtcac agtgaacttt tgctttagtt cactgctgta cagcactgag 51240 tgggaagata gttacaccct taaggatgat aatatatctt ctactttagg tgttctgctt 51300 ccatcttctt tcttctagaa aacatttttg tttaatgact cacaaaaaaa tatctgaaat 51360 gaatacagct ttatattttg cagttgtaaa aatgctaaac tagtataagg taattagctc 51420 tttcaaaaat aagtcgcttt ctacataaaa gaatgattaa atgaatagtt atcttctttg 51480 aaatttttta aatccacagt tacaggtttt atgggttttt ctttttttct attattttct 51540 tttttggtag caagccttat cttcacattt taaaaggttg tgttagaaag ccagtttctc 51600 tccttctccc cctacctcct cctccaccta gattcaacca cttcttatgg caaagagaat 51660 aactttctgg gctagagagt atgggttgtg tagtaaagat ttttgttgtt tttttgtttt 51720 ctcctcctac acttgacagc caacaaggct acaagtcttc tttggaagaa ctgcaaaatt 51780 cttgaattta ggacacagtg gtatcttttc tagaagtaca gagtcctgct ctgtgccttt 51840 aataccctaa gaacaaaaat catttttcct ctttaaggca agatctactg tgggtgatgg 51900 ctaccaccta cctctctaga cctcacagaa tcttcttctt aatgggcatc tctttttcca 51960 cccaaatcct ggcctttgaa tcagcatctc tcagagacag cacctctcac attgctcccc 52020 aagttcactg cattggagcc tgcagccaac tgatgtcctt ctcatcagtc tggttgttta 52080 ccctaaacac catctcacac atgctagctg gggatggtcc tcagactatt cattctcatt 52140 tctttgctta aaatttttct gtgtttgtat gggatgtctt ttatggtttg ccaactagaa 52200 cttttcagta gtatctacaa gcatttatta ttttctagac tcagtaaaaa gttagaattg 52260 aggggttcaa atatctcctc cattgtagtt gactttattt gaatgattaa gacatataca 52320 tttaaaaaaa tcaattctag tatagttttg gtttttggaa aatacctgat ttcactttat 52380 atttcctgtt gtctagcatc tttgttttct ttgaagtata tacaaaggca cactgtgttt 52440 tgatctggaa attggaggtg aatcctgaaa aatggaaggc aagcactatc agtgctataa 52500 aggattgtta aagtgtagtg ccagtggact gatcccagca ctgcgcagtg gtcattatca 52560 ttttggtaat ctgaagtaga agcatgcctc tagacatcct tccataattt tatataggta 52620 gtgtaaggat gactgtctat aaattcacct gaactacaga gagacttctg atataataaa 52680 agaaaaatac agagattgtt caggatggga atacaaacac taggaaactg gaaaagcaaa 52740 ttaaaacttc tgaattaata gaaaaagtta aataggaaag agaaaataaa gggaaacttg 52800 accaaggtag caaagaaaga aaatggaaaa gaggtaatta tgaggcagta taaaacaaac 52860 tctgtcgcaa aaaaaaaaaa aaaaaaaaaa aaaattccat ttgtagtttt aaaaaaatag 52920 taaagatata gaaaaattag tagaaaatac aaaaatctgt attcttcttt tatcagaatt 52980 aaataattat gcctgcccta ttttaccaaa ttattttatt aaaacatgct aaagaagttt 53040 ataagctttt cttctccctg ctcccattct ctataggatt tttgctgaga tatttcaaaa 53100 tttttatttt taggttatat ttaagctttc acttctaatc tctctcaaaa acctttcata 53160 tcacttgtca taaaaatttc ttctcatagt atcatcatcc agattcagtc acatatacat 53220 caagaataat tgtgcaatac tgtttcctag actcttcatc ttatcttttc aatgcatgat 53280 atttaaataa gccatacttt ttaatgtttt caatttttta atttatattt atttatttca 53340 aattttttat tatattttaa gttctgggat acacgtgcag aacgtgaagg tttgttacat 53400 aggtatacac gtgccatgat gttttgctgc acccatcaac ccatcatcta cattaggtat 53460 ttcttctaat gttatccctc ccttagcccc ccaccccccg acagatccct gtgtgtgaca 53520 ttcccctccc tgtgtccatg tgttctcatt gttcacctcc cacttatgag agaacatgca 53580 gtgtttggtt ttctgttctt gtgttagttt gctctgaatg atggtttcca gcttcatcca 53640 tgtccctgca aaggacatga actcatccct tttgatagct caatgttatt gcatggtgta 53700 tatgtgccac attttcttta tccagtctat cattgatggg catttgggtt ggctccaaat 53760 ctttactatt gtgaatagtg ctgcagtaaa catacgtgtg catgtgtctt tatggtacaa 53820 tgatttataa tcctttgggt acatacccag taacgggatt gctgggtcaa atggtatttc 53880 tggttctaga tccttgagga atcaccacac tgtcttctac aatggttgaa ctaatttaca 53940 ctgccaccaa cagcgtaaaa gctttcctat ttctccacat cttttccggc atctattgtt 54000 tcctgagttt ttaatggtca tcattctaac tggcatgaaa tggcatctca ttgtggtatt 54060 gatttgcatt tcgaccagtg atgatgagct tttttttcat gtttattgcc cacataaatg 54120 tcttcttttg agaagtgtct gttcatatcc tttgcccact ttttgatggg gttgtttttt 54180 gttttttttt ttgtaaattt gtttaaattc tttgtagctt cttgatagta gtcctttgtt 54240 agatggataa attgcagaaa ttttctccca ttctgtaggt tgcctgttca ctctgatgat 54300 agtttctttt gctgtgcaga aggtctttag tttaattaga tcccatttgt caattttgtc 54360 ttttgttgcc attgcttttg gtgttttagt catgaagtct ttgcccatgc ctatgtcctg 54420 aatgatattg cccaggtttt cctctagggt ttttatggtt ttcagtctta catttaagtc 54480 tttgatctat cttgagttaa tttttgtata aggtgtaagg aaggggtcca ctttcaattt 54540 tctgtatatg gctagccagt ttccccaaca ccatttatta aatagggaat cctttcccca 54600 ttgctttttt ggtcagattt gtcaaagatc agatggtaga tgtgtggcat tatttctgag 54660 gcctctgttc tgcttcattg gtctatatat ctgttttggt accagtacca tgctgttttg 54720 gctactgtag ccttgtagta tagtttgaag tcagctagcc tgatgcctcc agctttgttc 54780 tttttgctta ggattgtctt ggttatatgg gctctttttt ggttccatag gaaatttaaa 54840 gtagtttttt ctaattctat gaagaaagtc aatgttagct tgttggggat agcattgaat 54900 ctataaatta ctttggtcag tatggccatt ttcatgatat tgattcttct gatccatgag 54960 catagagtgt ttttccattt gtgtcctctc tgatttcctt gagcgttgat ttgtagttct 55020 ccttgaaggg gtccttcaca tttctcgtga cttgtattcc tagctatttt actccttttg 55080 tagcaattgt gaatgggagt tcagtcatgt ttgggctctc tgtttgtcta ttattggtgt 55140 ataggaatgc ctgtgatttt tgcacattta ttttgtatcc tgagacttca ctgaagttgc 55200 ttatcagctt aaggagattt tgggctgaga tgatggagtt ttctaaatat acaatcatgt 55260 cctctacaaa aagacacagt ttgacttcct ctcttcctat ttgaatacgc tttatttctt 55320 tctcttgcct gattgccctg gccagaactt ccactactat gtttaatagc agtggtggga 55380 aaggacatcc ctgtcttgtg ctggttttca aagggaatgc tttcagcttt tgcctattca 55440 ttatgatatt cgctgtgggt ttgccataaa tagctcttat tattttgaga tacggtccat 55500 caatacctag cttattgaga gtttttagca tgaaggggtg ttgaattttg ttaaaggcct 55560 tttctgcatc tattgagata atcatgtggt ttttgtcatt ggttctgttt atgtgatgga 55620 ttacgtttat taatgtgcgt atgttgatcc aggctttcat ctcagggatg aaactgacct 55680 gatcatggtg gataagcttt ttgatgtgct gctggatttg gattgccagt attttattga 55740 ggatttttgc atcaatattc atcagggata ttggcctgaa attttctttt ttgttgtttc 55800 tctgccgggt tttggtgtca ggatgatacc aaataaaatg agttagggag gattccctct 55860 ttttctattg tttggaatag ttttagaaag aatggtgcca gctcctcctt gtatctctgg 55920 tagaattcag ctatgaatct gtctggtcct ggggtttttt tggttggtag gctattaatt 55980 actgcctcaa tttcagaact tgttattggt ctattcagga attcaacttc ttcctggttt 56040 catcttggga gggtgtatgt gtccaggaat gtatgcattt cttctagatt ttctagttta 56100 ttttcataga ggtgtttata gtattctctg atggtagttt gtatttctgt gggatcactg 56160 gtgatatccc ctttatcatt ttttattgtg tctacttgat tcttctctcc cttctttatt 56220 agtctggcta gtggtctatc tactttgtta atcttttcaa aaaaccagct cctggattca 56280 ttgatttttt ttttttaagg gtttttcatg tctctatctc cttcggttct gctctggtct 56340 tagttatttc ttgtcctctg ctagcttttg aatttgtttg ctcttgcttc gctagttctt 56400 ttaattgtga tattagcgtg tcaattttag atcattccca ctttctcctg tggacattta 56460 gtgctataaa tttccctcta aacaaacact gctttagctg tgtcccagag attctggtat 56520 gttgtgtctt tgttctcatt ggtttcaaat aacttattta tttctgcctt aatttcatta 56580 tttatgcagt aatcattcag gagcaggttg ttcagtttcc atgtagttgt gcagttttga 56640 gtgagtttct taatcctgag ttctaatttg attgcactgt ggtctgagaa actgtttgtt 56700 attatttcca ttctttcaca tttgctgagg agtgttttac ttccaattat gtggtcaatt 56760 ttagaataag tgcaatgtgg tgctgagaag aaggtatatt ctgttgattt ggggtagaga 56820 gttctgtaga tgtctcttag gtccacttgg tccagagcct gagttcaagt cctggatatc 56880 cttattaatt ttctgtcttg ttgatctgtc taatattgac agtagggtgt taaaagtctc 56940 ccccattaat gtgtgggtgt ctaagtctct tcataggcct ctaagaactt gctttatgaa 57000 tctgaatgct cctgtattga gtacgtattt ttaggatagt cagctcttct tgttgcattg 57060 atccctttac cattatgtaa tggccttctt tgtcttttga tctttgttgg tttaaagtct 57120 gttttatcag agactaggat tgcaacccct gcctttttta gctttccatt tgcttggtaa 57180 atattcctcc atccctttat tttgagcata ttgtgtctct gcctgtgaga tgggtctcct 57240 gaatatagca caccaatggg tcttgactct ttatccagtt tgctggtctg tgtcttttaa 57300 ttggggcatt tagcccattt acatttaagg gtaatagtat tatgtgtgaa tttgatcctg 57360 tcattatgat gctagctggt tattttgccc attagttgat gcagtttctt cctagcatcg 57420 atggtcttta caatttggca tgtttttgca gtggctggta ccggttgttc ctttccatgt 57480 ttagtgcttc cttcaggagc tcttgtaagg caggcctcgt ggtgacaaaa tctctcagct 57540 tttgcttgtc tgtaaaggag tttatttctc ctttgcttat gaagcttagt ttggctggat 57600 atgaaattct gggttgaaaa ttcttttctt tagaaatgtt gaatattggc ctccactctc 57660 ttctggcatg tagggtttca gcagagagat ctgctgttag tctgatgggc ttccctttgt 57720 gggtaacccg actttctctt tggctgccct taacgttttt tccttcattt caatcttggt 57780 gaatctgaca gttatgtgtc ttggggttgc tcttctcgag gagtatcttt gtggtgttct 57840 ctgtatttcc tgaatttgaa tgttggcctg tcttgctagg ttggggaagt tctcctggat 57900 actatcccga agagtgtttt ccaacttggt tccattctcc ccgtcacttt caggtacacc 57960 aatcaaacgt aagtttggtc ttttcacata gtcccatatt tcttggaggc tttgtttgtt 58020 ccttttcatt ctttattctc taatcttgta ttcatgcttt atttcattca gttgatcttc 58080 aatctctgat atcctttctt ctgcttgata aatttggctg ctgatatttg tgtatgcttc 58140 acacagttct tgtgctgtgt ttttcagctc catcaggtca tttatgttct tctctaaaat 58200 ggttattcta ggtagcaatt cctctaacct tttttcaagg ttcttagctt ccttgcattg 58260 ggttagaaca tgctccatta actcagagga gtttgtcatt acccgcctcc tgaagcctac 58320 ttctgtcaat ttgtcaaact cattctccgt ccagttttat tcccttgctg gtgaggagtt 58380 gtgatctttt ggaggagaag aggcattctg atttttggaa ttttcagcct ttttgcactg 58440 gtttttcctc atcttcatgg atttatctac ctttggtctt tgatgttggt gaccttcaga 58500 tggggttttt gtgtggacat cctttttgtt gatgttgatg ctattccttt ctgtttgtta 58560 gttttccttc taacagtcag gcccctctgc tgcaggtctg ctggagtttg ctggaggtcc 58620 actccagacc ctgtttgcct gggtatcact agcggactct gcagtacagc aaagattgct 58680 gcctgttcct tcctctggaa tcttcatccc agcagggcac ccaccagatt gccagccaga 58740 gctctcctgt gtgaggtgtc tgtcgacccc tgctgggagg tatctcccag tcaggaggca 58800 caggggtcag ggacccactt gaggaggcag tttgtccttt agcagagctt gagcactgcg 58860 ctgggagatc cgctggtctc ttcagagcca gcaggcagga acgtttaagt ctgctgaagc 58920 tgcgcccaca gccacccctt cccccaggtt ctctgtccca gggagatggg agttttatgt 58980 ataagcccct gactggggct gctgcctttc tttcagagat gccctgccca gagaggagtc 59040 tagagaggca gtctggctac agcagctttg ccatgctgca gtgggctctg cccagtttga 59100 agttccaggt ggttttgctt acactgtgag gggaaaactg cctactcaag cctcagtaat 59160 ggtggacgcc cctcccacca ccatgctcca gcgtcccagg tcgacttcac actgctgtgc 59220 tggcagcgag aatttcaagc cagtgaatct caacttactg ggctctgtgg ggatggtatc 59280 tgctgagcta gatcacttgg ctccctggct tcagcccccc ttccagggga gtgaatggtt 59340 ctgtctcact ggcattccag gtgccactgg ggtatgaaaa aaaactcctg cagctagttc 59400 attgtctgcc caaatggctg cccagttttg tgcttgaaac ccagggccct ggtgatttag 59460 gcacccaagg gaatctcctg gtctgtgggt tgcaaagacc atggggaaag catagtatct 59520 gggccggaac acattgttcc ttacagcaca gtcactcacg gcttcccttg gctaagggag 59580 ggagtttccc cactctttgt acttcccggg taaggcaatg ccccaccctg cttcagctcg 59640 ctctccatgg gctgtagcta ctgtctagcc agtcccaatg agatgaggtg ggtacctcag 59700 ttgaaaatgc agaaatcacc caccttctgt gttgatcgcg ttgggagcta cagaccggag 59760 ctattcctat tcagccatct tgccagccag caaccactct actttttttt tttttaattt 59820 aagttctggg atacatgtac agaatatgca ggtttgttac atagatatac atgtgccatg 59880 gtggtttgct gcacccaaca atccatcatc tacattagat gtttctacta gtgctatccc 59940 tcccctagct ccccacccct caacaggccc tggtgtgtga tgttcctctc cctgtgtcca 60000 tgtgttctca ttgttcacct cccacttatg agagaatatg cagtgtttgg ttttctgttc 60060 ttgtgttaat ttactaagaa tggtttccag cttcatccat gtccctgcaa aagacatgaa 60120 ctcatccctt ttgatggctg catagtattc catggtgtat atgtgccaca ttttctttat 60180 ccagtctctc actgatgagg atttgggttg gctccaagtc tttgctattg tgaatactgc 60240 tgcagtaaac atacatgtgc atgtgtctta tggtacaatg atttataatc ctttgggtat 60300 atacccagta atgggattac tgggtcaaat ggcatttctg gttctagatc tttgaggaat 60360 catcacactg tcttctacag tggttgaact aatttacact cccaccaaca gtgtaaaagc 60420 tttcctattt ctccacatcc tctccagcat ctgttgtttc ctgacttttt aatgatcgcc 60480 attctaactg gtgtgagatg gtatctcatt gtggttttga tttgcatttc tctcatgacc 60540 agtgaagatg agcttttttc catgtttttt ggccacataa atgtcttctt ttgagaagtg 60600 tctattcata cccttcaccc actttttgat gttttttttt tcttgtaaat ttgtttaagt 60660 tccctgtaga ctctggatat tagccctttg tcagatggat aaattgcaga gattttctcc 60720 cattctatag gttgcctgtt cactccgatg atagtttctt ttgctgtgca gaaggtcttt 60780 agtttaatta gatcccattt gtcaattttg tcttttgttg ccattgcttt tggtgtttta 60840 gtcatgaagt ctttgcccat gcctatgtcc tgaatggtat tgcctaggtt ttcttccaag 60900 gtttttatgg ttttaggtct tatgtttaag tctttagtcc atcttgagtt aatttttgta 60960 ttaggtgtag ggaaggggtc cactttcagt tttctgcata tggctagcca gtttccccaa 61020 caccatttat taaataggga atcctttccc cattgttttt tttggtcagg tttgtcaaag 61080 atcagatggt tgtagatttg tgacgttact tctgaggcct ctgttctgtt ctattgttct 61140 atatatctgt ttgggtacca gtaccatgct gttttagtta ctgtagagtt gaagtatagt 61200 ttgaagtcag gtagcatgat acctccagct ttgttctttt gacttaggat gtcttggcta 61260 tacaggctct tttttggttc catatgaaat ttaaagtaat ttttttctaa ttttgtgaag 61320 aaagtcaatg ttagcttgat ggagctatta ttggatctat aaattacttt ggtcactatg 61380 gccattttca aaatatacat tcttcctatc tatgaatatg gaatgttttt ccatttgttt 61440 gtgccctgat ttccttgagc agtgttttgt agttctcctt gaagaggccc ttcacatccc 61500 ttgtaagttg tattcctggg catttaattc tctttgtagc aattgtgaat gggagttcac 61560 tcatgatttg gctctctgtt tgtctattat tggtgtatag gaatgactgt gatttttgca 61620 cattgatttt gtatcctaag aataagccat actttttaat atattttatt cataaaaaaa 61680 tgcacataaa gcaaatgaca tatagaaaca atatgattaa catccacctc tggactcagt 61740 tggcttaaga aaatatgaat attatcacta cacttgaaat tgattgcata tttcttcctt 61800 atcacattcc tctttatctt tctccagaaa cctctatatc tttaatttgg agttggtcat 61860 tttcttatat tattctgtag gattttttaa tatctatgta tttctcacta atagattttt 61920 tagttttgag atgttttttt ctactccata gaaatgctat gtatatttat gcaatttgat 61980 cttactgcct aatattatat ttgtgttttt tattttgatt tgtattattc tggtttgttt 62040 tttcctacac tgtatgattt cattgtatga gtatctccca tttatttatt ctcatgtatt 62100 gacatgtact tccacttgat atttgctgtg ctttacctac agttcaattg tgaacattat 62160 tttacatgta ttctggtggt tcatgtgtga gagtttctgt aggtaacctg cacacctggg 62220 catgaaattt ctgactgtaa tacatgctca tgctgggctt tactagataa tgctagattg 62280 tttttaaagt agtagtagtt tacactccat ggcagctctt cattgttttg tgtgttcctg 62340 tattctgtgt ccttgccgtt ttaggtagat gatgtctcat gtggtcttac tttgcatttc 62400 tctggttaat aatgaggtca gtcatattgt tgtattttgt catctatata tttcttcctc 62460 tgtgaaatat ctgttcatgc cttttctttc tgatttatag gaattctttg tgtattttgg 62520 gtatatatgt tataaatatc tataggatag ataaaaacaa gctatctgat aaggcgatat 62580 gcagaaatat aaattaggtg aggaagcaag ccatgcaaat atccaaggaa aaaatcattt 62640 catgcagagg aacttgtcag tgtaagaccc caaggcagaa acacacttca agggcaagat 62700 gggcatcaat atgggtggag catgagggag aatagaagag agaccatcta tgcaagggta 62760 agttgggcct tgtagagctt gatatgaatt ttaacttcat tctaagtagg atcaggagtc 62820 actgaaaata tcaagcaaga atgacatagt ctgattaagt ctttaaagtt gtatagaaaa 62880 tagtctacaa aaagtgaaag ctgggaaacc agccagcata tgttacaata gtccaactgt 62940 gattgtgggg acataaatta tggtgcaagc agtggaaaca ggtatttgga tttgggttat 63000 attttcatgg ttatccacta ggatttgctg atgtattaga tatgaggtga gaaaataagt 63060 gaggtatcag taatgactct agagtttggg gctagagcag caggtgtgta aatgaagtta 63120 caaacctaga agatgcatgt ttggaccaat gcatcaaagt tctgctctaa acatattaat 63180 ttgaggggtc tgttagaaat acaagtggag atgctgagaa tacagttgca tagttgagtc 63240 tagagtttct tggaaaagct gggtctagga ctgtaaattt ggtaatcatt aatgtctaga 63300 tgatattaga gacatagaac tagatgcaga cacctagaaa gagcatagat atctaagaga 63360 agtctgagga ctgagcttgg tgtactcaaa gatttagaag ttgagaagat gaaaaagagc 63420 tagacagaga gactgaaaga gtgactacaa gaatgaaata tgtggaagcc aaatgaataa 63480 gagcgttaaa agaagaagga aacgatcaat gtgttaagta cttacccggg ttaagaccag 63540 gactgagaat tggccattgt tttttgacat ttagaaatct ttgaaggcct tgccaagaat 63600 aatttggttg ggataaggag aggaatgacc tgactggaga aggttcaagg gagaattgga 63660 ggagaggaaa tgaagagagt gatatttctc cacggaatac attatgtgtg agagggacta 63720 ttagaggatg cagacaagag ggcaaatgat ttcagagaaa agtccctgag tgggtgagaa 63780 ggactaagat ttggtattga aatgtcagga gtatgaattg taacaggagg gaaggcagag 63840 tacaggggta gccatactgt agattggcca caaagaaaat attctccaaa tggagcttct 63900 ttccccctaa gcctccccca aatcattacc tctgcttctg attcaatccc tcaaaacctc 63960 atccatcagc ttgaatgaga gacaactatt cagccaccat gtccatccgc tttcttatcc 64020 ccccttgctc acccccagcg tctcagccca aatcttccct ccttccagcc aacaaagctc 64080 ccatgcctgt catctcctga tgtcaatgca gcgggcaggg gagtggggga ccactgaccc 64140 cacatgccta ttctctgctt agattaccca aaataccagg tgtcgttatt tttcagaggt 64200 gcaaaaatat aatacaaagt cattctaaac tgtcactgaa ttcagagtac tttttgccat 64260 tgtctagtca ctgaattcag agtacttttt gccattgtct tattttctca atcaacaaat 64320 gctaagagta caattatatg aagttctaca gatttatttg ttacttgcat tacttagtaa 64380 tggttcttat gcataaaggt taggaataac ttctttgaac aaatatccta tataaaaaaa 64440 caacaaagtg gtttttaaaa atcacattgc tgttcacatt ccctttttag tactctcttc 64500 tcatattctt ttttaagcta tgttaggtca aaacaactac tttaataact tagcaaatat 64560 ctatcaggag tccactgtgt agttagcagt ttataaagct tcagatctac attagtgagt 64620 gaaatggacc tagcccctat ccttacaggg cagcaagcct ggtggaggaa gctatgttct 64680 ctgaaggcaa caaaaacagt catgcgtcat ttaagagtag ggatacattt tgagaaatgt 64740 gtcatgaagt agtcaatttc atcattgaac atcatagaat gcatttacgc agacttagat 64800 ggtatagcct actacacacc taggctgttt ggtgtagccc attgctcctg ggctacaaac 64860 cgatacagca tgttactctg ttgaattttg taggcaactg taatgcaatg gtaagtattt 64920 gtgtacctaa acatatctaa agatagagac agtacagtaa aaaatactat cttatgaaaa 64980 cactgttgta tatgtggtct gtcattgact gaaacaatat tatgtagtgc aaaccataca 65040 gcaacagata ataaagacaa gatattcaga gtgggagatg gaaaagacaa taatgtattc 65100 cttcattcat ttagcagaca tgtattgcca aaagacactg gaaaaaataa ataaaggtga 65160 caaacaggtg gtgcctttgt tgaagattat agtcatggac agggaaactt tagccctctt 65220 cttaacatta tcttccctta gacacaaaca cacacacaca caattcctgg catatgctat 65280 atactccata actgttcctt cccttccctt tgtgactcat tccttccccc agaaagctca 65340 tttgatctct gcaaataatt ttgaatcccc tttgtttttt aaccatggca attgttttgt 65400 tttcccatct cctacaagtt ttagctcaac tcgttggggc tgtttttttt tttttttttt 65460 tttttttttt tttttttttt ttgaggtgga gtttctccct tgtcacctgg gctggagtgc 65520 aatggcatga tcttgactca ctgcaacctc tgcctcctgg gttcaagtga ttttcctgcc 65580 tcagcctctg gagtaactgg gattacaggt gcccaccacc acacccagct aatttttgta 65640 tttttagtag agacggggtt tcaccatgtt ggccaggctg gtctcgaact tctgacctca 65700 ggtaatctgc ccacctcggc ctctcaaagt gctagaacta caggcatgag ctcaccatgc 65760 ctggccattc cttggggctt tatagaacaa gtataatcct ttttctttat gacaataccc 65820 tgagttcaga aatctcagag acaaatgttt acagagacca ggtaaataag tagctagagt 65880 gacaagactt aagaaaagag accattggcg taccaccagt ttgtggaggg aactggaaaa 65940 actggaatac acatgcccca tccaaaagca accattgcaa ctaaacttta acagattgtt 66000 gccacctaag taattcacgg atggtctcat aattctggtc agcattgtct gagccaaaca 66060 aaatgtatct atgggcatga tcagatacta gagccagcag attgcaacct ctgcttagat 66120 aattgcaggt atcagccttc ccttggctaa acagctactt catactgata agtagccctt 66180 gcctggcaca aagcaggtgg ggctgaatcc agcctgatat cacatcacca caactttctc 66240 taattctcct caaggcgtct gtgaactacc agcagcccaa ataagcatcc ttttctcctt 66300 caatcatttc tcataaagca gattctactc atctcaccat ccatcttttt cccctaacat 66360 atcagaattc atcaaagtag aaactccaga aatcatccag aaatacaaat gtgatctgac 66420 cagcacgggt ataaactttc tctttcctta atttagaaaa aggattatta attttgagtc 66480 ttagatctca taaaagtaga gctggattga ttttaaggga tcaatgaacc tactgaaatt 66540 gtataaaaag attttgtggg gctggcaaac aaatgctttt cttttaacat attcttaaaa 66600 gaacaaaacc tcaaagaaga tttaacaacc actatagact aagtttcaat taatgtagtc 66660 tgtagcatta gattttggaa ggctgttcta tcagaatatg gaccaatact gagcgtagtc 66720 gctactaaaa ctctttgtct ttttattcat atcttctcct aaaaagctcc atgacccctg 66780 cagaatactg gtataattac ccacacctca cccacccagt gattgttgat gtaactaatt 66840 atttaatcca agtctgaggc ctaaattttt tttaatcaag agttagttac atgttttaat 66900 ttattttttg taattttaat atctaacact tttattctat ttcattcgat tgatgctaaa 66960 atgcataatg ctatgattct aagacttagg aaaaataata ctatactggg atatggagtt 67020 tatatatatt tacatgaatt tctttttttt cttctctgta ggtcgaaaat ttaaaaagtt 67080 caataaagtc agagttgtga gagcactgga tgctgttgct ctcccacagc cagtgggcgt 67140 tccaaatgaa agccaagccc taagccagag attcactttt tcaccaggtc aagacataca 67200 gttgattcca ccactgatca acctgttaat gagcattgaa ccagatgtga tctatgcagg 67260 acatgacaac acaaaacctg acacctccag ttctttgctg acaagtctta atcaactagg 67320 cgagaggcaa cttctttcag tagtcaagtg gtctaaatca ttgccaggta ataataattt 67380 ttatatacag catgtaataa aaaatatact atgtttgttg tattagtaca ttgttaaata 67440 aaacactcta tgcagtaaat taactacaat ataaaacagt atgtgatagt atgtgtttga 67500 gcagttcaaa atatttgtct aggaccctaa tatgtgttac agaaatataa aaaattagag 67560 aacacactga gtcttactga tgagtcagaa attgcataca ttaaacttat gctaataaga 67620 aatagcttag gtttatgtgt tcaaccacag atattagagt agctcagaaa tgataaatgg 67680 accatttttt ataaatgccc tatttattct aacccttaat tcaaccaacc tcatttggtt 67740 gaaaataaaa ctgaatgtat aaagtcatat aacaaatcaa agtagtattc agagtacagt 67800 aaggtgtcaa aagtaaatta agaaaacaaa attccctaac gtaaattact ttaaaaaata 67860 atagcacaca gagaaaagaa tctctagtca ctattcctct aatttatacc caaagagagt 67920 atatcgccca gcaacagaga gaacttggat tatatcatca cagaagaatt tcagtcactt 67980 tacttatcag ctatttttgc tccctcagca cagctctgta ggagccaagt acttgaaaga 68040 ataacaagga tgaaattctc acttggttaa atcttagact gcagagccag gagaatacat 68100 gtatagcaat tgtttgtgaa ggcattactc ctataattag aaatagaact ttttaaaaaa 68160 ctacattctc ttcacatcag taataccctt atactgctat agacaaatta tttttctaat 68220 atctgatcaa gaattaaaca tattatgtcc ttttagtccc caagtaaatt caatggcact 68280 tttagaaaaa gaatttcttc ccaattctcc ttaatataat tctcagttat tattctacct 68340 aaaaggtttg aaatacaata gatgaattgt tactgaaaat tcaattacat gaaacagaaa 68400 gaaaaataga tacctgtttg ttgaatcttt caaaaaatac atattaagaa aagttaatag 68460 aattcagtat tctagtggag atgaagtaat tttatagaaa aatcaaaatg cccagattct 68520 tactggcttc agtctcacca aaaggcctta aacagacaat aattttttga acattctaaa 68580 tgtttaagat ttatcacata gtggttatca aacttgacag agctttaata aataaaaata 68640 acccaatagc attgatgaac tttttctgtt taaaaatgag tgtttaaaca tgtgtttaat 68700 ttttctccat tgttctatta tttggaaaat atgatttctt tggtttttgt tgatatagtt 68760 tatattcctt ttgtttgcaa ttttggaggt tctttactat ttaaaaagat tctagcatca 68820 gctggtaacg ttagaaaata accagttata cttgaaaaga tgacttattc actttggata 68880 aaagctaagt gaccttttta accggaaacc gttatctatc aggacttttt gacccccaca 68940 caaccacaaa aaggaatgag agaaaactct tacattgaat ttggtggcaa cttttttaat 69000 gctgcaaaat gtaatttttg tgtagtaaat cagaataatg gcaacactag tattacccat 69060 actgtttcca ttaattgcaa acagggaaac attgagatta tcaaagtctc ttgtggcctg 69120 tggactttta aagccatatt ttcatggtct gcattttggg aatagcacta ttgaatgaat 69180 tatgaaaatg gtcagtgagc tagagtgact atttgacctc tgagaaagga cttggatgga 69240 cacatgtata gaaagtactt tagtattatt agctacttaa aactgatttc aattgacttg 69300 aaataagctt acagttttta ttcacttatt cactcaataa atgtctatta tgtacctgct 69360 gtgttcaata tctaataatt atacagtggt ttagaaagaa aagccacaag gctgggctca 69420 tggtgtggat gcattttggt acagcacatg ctagcatctg gtcatcagtt ttgcatgtaa 69480 aagaaattta tttcgttcat tcactcatgt gtaataaaac taataagtct gtggaagaca 69540 gacttttgtt aagtgatcct cactaattag ccagaatgtc aactaacttg cttatttaca 69600 gtgagacaca cacacacaca cgcacacaca cacaatataa atatacataa tacttatgtt 69660 gctaggagaa tatattaaca gatcagatca ttctttcaca ataggaaact gtatttggaa 69720 agtcacatta agatctgatg cagaatgacc atcataacaa aggtgacagc gtttatgaag 69780 acacctaggc atatatttgc aggtgcccta agaattctta ttgcctggta gggctttctc 69840 atttcttctt tgtctacact cttccattat cttggatctg ttataataat ggtagtttat 69900 atttcttcaa aaatatttca gatatatcct tttgtttctt ttcttttttt tctttttttt 69960 tttttttttt tgagagaggg tctcactcta ttgcccaggc tggaccacag tggcatgatc 70020 ttggctcatt gcagcctgga cctcttggtc tcaagttatc ctaccacctc agcctcctga 70080 tgagctggta gctgggacta caggcatgcg ccaccttgct tggctaaatt ttttttattt 70140 ttagttgaaa ttgggtttaa ctgttttacc caggctggtc ttgaactcct ggactcaagt 70200 gatctgcctg cctcagcctc ccaaagtgct ggaattacaa acatgactca ccgcacccca 70260 ccccttttac tttaatagag acttgcaaga tgctctagcc ttatgtcagc acgtttataa 70320 accatctctt aggatacatt gcaaatgagt gattttgcat gggcttttat ctgtagcctt 70380 agtgggcaat attttcttat ttgaaatgta gagcaacacg atatttgaat ttttttaatg 70440 aacaagacat attccctcta ttaaggaact ctcaatccag tagcagagag aagataccca 70500 aacaactata gtgttatgtg aaagtgatat aagatacaga tataatgaaa gtggggacag 70560 aagaacattt ttggcttcaa ggactaaaga agccttcaga gacaaaaatg tcagttaaac 70620 tgcatcttac aggagaaaca agactttgat aaatcatgat aagagaaagc attctagata 70680 gaaggaacag tatggctgaa ataagaaaag catgagatag gtttgctgaa taatgcatta 70740 aaccagcata ttaatggttc tcaacctttt caagtgtggg gggtccctta gtaacatcaa 70800 aaattttata gtctccccat agggtagtaa ttaaagcttt tagtagctat ggattgagaa 70860 aacacatttg tacatacagt ccctcggaca ctgctacagt aagtactggc cttcgggaga 70920 tttaatcacc caaatggtaa gaaaccattg aaaagaaatt tattttgaac tgaataatgg 70980 aaagtaaact tagaggtgta agttataggc tgatcaagga gggtcttaaa aattactgta 71040 gatgttagag tttaaattaa aagtactaat tcacaattcg gtgtcacctt taaattgtcc 71100 tcatcctgta catcaatacg ttgcagtgaa attgatgtga cagagggggt atgagggaaa 71160 cacttccgtt gcataaattt tgtcttgata tttcagttaa cttagtgaaa actccattta 71220 ggtcaatgct ttgaattttt aaaccttgca ttttttagga catactttat tctttgtgtc 71280 atttcaccag actacttgca attatcagta tcagaatatt agaacagtat tttgcctcat 71340 ttttctgctt ttgaagcgtg ccaaaacaac ttttgagtca gtgagtcatt gcatcttgac 71400 agaggatttt ttctcttcct tcccgaatca gaagactaaa ttaaattatt gctctccaga 71460 aaaaaatgat gagagagaaa tgagagtagc atactgagag taaaggcctt aataaatgtg 71520 ttgataaaga ctaaaccatg gtttttctca caccctattc cactgaaatt atgattgcca 71580 gtggcaacaa aaatttttac ttgtcaaatt agatgaatat ttccagccct taatgtgttt 71640 cacctacctc tgggactatt ggcaaagtaa ctatttcttg ctaagactct tgcttaggta 71700 tttgtcactt cccttccagg tagatatgtt tctctcccta tgtacatttc ccgactcctg 71760 tgtggatttg ttcttccacc cacactataa atttgtttgt gttctgggtt ctatcttttc 71820 cagtcatttc tcactctaca cagtctgcat aagctcattc atagtcttgt cttcaactac 71880 cactatattt cgatgactcc taaatacata atattagcca gaccctctac aaaactactg 71940 aactttcaac tgcttattac acagctccac ccagatgact tctaagtcag catgaccccc 72000 taaacctaag gccttttcca gtatcttgaa atggcacctc agcaagaatt tttggtatca 72060 tccttggctc agtttctcct tcacccctta cccaataaat tgtcaaattc tatcaactcc 72120 acttattaaa tatatgattc attatacatg ctacttctgt tgctacttcc ctgacttagt 72180 ttggtgctgg tcttttttta cttggattca tgttgcagcc tcctgactga ttctattacc 72240 atcaatttca cgtggtgccc aaatgatcct ctattctacc cccagaatca tttttctaaa 72300 acaattttaa tcaatcatat tttccatttc tttttccaaa tagcatccaa actccatggc 72360 ctcaatatcc aagggctttc tgtgatctgg ctcctgaatt tgcagtgtta atctctcatc 72420 attcctcttc acacgactct aagatctagt tccctaaatg ctgtttgctc aaattctagc 72480 cttcttctct tgcccatatt atttttccta cctggcagcc catctctttt cccacgtctt 72540 cctttgactg acctgtaact atctttcagg attcgcatta cttgtcactt gctctgggaa 72600 gatttctctg agccttccta gactgggtga ggaaaccctg taccctccct tgtctccttt 72660 gtgcttgtca accataacac tatgactctg agttacaatt gtgttctact tttctatctg 72720 tctcacaagc ctatagtgtt aaaggcagaa acagcatctt tctaaccttg atcctaattg 72780 caagtataat gcttcctccc aaatagatgg ttaaagaata tttatagaaa tttaaattac 72840 agaattgatt tgcaaagaac tgtactacct tagattatta gacattaact tcttattcag 72900 cataaatcat caaacatacg ttggtgatta tacttaatca tattgaagct tctttgtgct 72960 tctcaacatt gatacttgtg tatgaaagtt gttgctttgt gtcagaaggt gctgagatat 73020 ttaatgctct gccattgttg tagattatct gtttcaatta catgacattt tttacagaac 73080 caaaacctac atattttatc aaaactctgg gagtaagatt ggggaactta gtagtactgt 73140 catacaggcc atcttttttt tttttttttt tcttgagata ggctctcact ctgtcaccca 73200 ggctggagtg cagtggcatg attgtagctc acggcagctt agaactcctg ggctcaagcc 73260 atcgtcctgc ttcagcctcc ccagtaactt cagcctcccc agcactacag gtgtgcacca 73320 ccacacctgg acaagctttt taaattggtt tgtagagact gggtcttggc tggtctcaaa 73380 ttcctggcct caagtgactc tcctgcctcg cctcccaaag cactgggact acgggcatga 73440 gccaccacac ccagccccag gccatctctt cactatatac tttctgttaa ttctttaagc 73500 taaatttgga cagctgtaac agggcttttg tcagaaatta tcaggtcaag tcacttaaaa 73560 ggaaaagtgt tttttttttt aacatttgtt aaacacaatt agtgataatc catcacttct 73620 ccatgtgaag gtttttcatt ttggtcatct caaatgtact aagtttacct gatcattatc 73680 atcactggga atttaatctc tgtgttccca tctttgtttc agcataaagt aatgaatgat 73740 attacagtta ataaggaaaa aaatagcatt tctatactag gcatgttcaa gtcaatttaa 73800 cattgtacat ttatcacaga taagcactgt aaaaggtgga gtaaataaag gttattttct 73860 gagttctatt ccattctgtc aaacaaacag aaatatagtt tctgctataa atattggtaa 73920 ttttataaat ttaaagttat tgtaataaga tttgccttta atgttgtcat aaaataaaag 73980 taagcctgga tttctagtcc cagaatgcaa aaacaaagaa aaaagcaaaa agacgaaaac 74040 aaaaactaag agtaggataa agtgaatgta aacatgtaaa tgtagacagg atctgttttt 74100 ggataaagaa caatttaatt cttacataat tttgtaaaat gtttaaatgt ttaaaactac 74160 tcaaaggtta acataaaaca aatgcctgaa gtgaaaaatt acagcatagt taagtcaaat 74220 atttaaatat tataaaatac tttttttaga aagttataaa gttcaccaag aaagatatag 74280 agaataataa atgtatttgt ggagattatt gtgaacactt ggttaatgtt ttaatacatc 74340 tgattaatga aaacgaactt tctatgagcc tttttttttt tttttttttt tttttttttg 74400 agatggagtc tcgctgtgtc accaggctgg agtgcagtgg cacgttcttg gctcactgca 74460 acctcagcct cctgggttca aaccattctc ctgcctcatc ctcccaagta gctgatgaga 74520 ctacaggcat acaccaccac acctagctaa tttttgtatt tttagtagag acagggtttt 74580 accatgctgg ccaagatggt ctcaatctct tgaccttgtg atccacccac ctcggcctcc 74640 caaagtgctg ggattacagg cgtgagccac tgcgcccagc ccattttttt gtattatatg 74700 aaatatataa tttgttatat ataacaacac acataaaata tatattctgg aaaatatata 74760 ttccagaaca aacacataat ttatctttaa aattttttca ccattatttt ttatacttca 74820 gttctatctt atatgttcta taatatataa aatgcctcct aacacttttt tttatggcat 74880 tgtgttttag tattagagac ttcccattaa actgactggt ggatttcata ctagtattac 74940 tccaatgagc aattctgttg cattgataga ccaataataa atatctccat agtgttctac 75000 agtgtgcata attatcccat ttgtctcata gcaactcact gagttagtta tcattgtgaa 75060 ttgtgatttt tacttatgaa gaaatttaga ttcaatgata ataagtgatg tttccaaagt 75120 ggctgttagt aagtggtaga gccaggactg gaacttgggt cttctgtcct gaatgccttc 75180 caatttgttg agacagatca gcaataggga ggtttatatg tgcctccctt tagaaaggct 75240 tccttttcaa aaattaagac aattcattga tctcttgtca aatgattcta aattttatca 75300 caaaattacc catttttata gtctcttaaa tatttgactt acaagcagca tttatgtatt 75360 agatttattg tattgagctt tataattttc ttaaaaagtt attatgtaga cctatacttg 75420 aagacacgtt caagtcatat gcaaattaaa aaatgataaa acttgcatac caaaaatttt 75480 tttggctgct tagttagaga gtctttctgc acctgagtaa accaggattc ttggatgcca 75540 gtattaaaaa taaactatgt taattataaa acacacaagc aacaacaaaa aaccctgact 75600 tttaaaaaca aaagaaaaag aaaaaaaata atttactgag aggacaacag gggttcactg 75660 aatagaagct agaggacaag cttagaatat gggcagaagc caaggcacat ctgaagggct 75720 aaaaagcaag catcacaaat cctcccattg gttctcatta tctggacacc accaacatca 75780 agaataaata gtcatatctt tctctttgct ttagtatttt ctgattcaaa ttcctgttag 75840 tatatgttcc atgggccaaa cctatgcccc aagcccaaag aaggaggagg tttacccagc 75900 tttctccgtg ggaaagtagg aaggtaaggc atttgctctc gcaaaaacta caaccaatgg 75960 ggaactctgc ccacacagag aaaggaagga ggggaggaag gaaggaggga aggaaagaag 76020 gaaagcagga aggtattagg gagcagtgag ggaggaggag gaaaaagagg aaaggaagaa 76080 agagagggaa ggaagaaagg agggagggag gaagggagtg agagaagaaa ggagggagaa 76140 ggggaagaaa gaaaggaggg aaaaggggag ggaagaaagg aggaagaagg ggagggaaga 76200 aaggagggag aaggggaggg aagaaaggga gaaggggagg gaagaaaggg agaaggggag 76260 ggaagaaagg gagaagggga gggaagaaag gagggagaag gggagggagg aaaggtggga 76320 aggagaaaag gttagacatt gaagagcatc ccttctcagt aaaaggtaaa tgctcactat 76380 ggcatctcta attatgaata tgcatttgag tttttggtct tattgaggct aaggagttct 76440 aagaagagag tagcaatttt tctactattc acatgaagat gggacgatct gaagtgtatg 76500 aagagaagag ccttcttttt tgctacctac cttgacatga ttaacattgt caggaaataa 76560 ctttcttttg ctcttgatag ctaatgaatt aagttttgct ttggttttac ttttgaaaca 76620 gattatttgt aagaacatca gtggattttt agagaaaata gatgttttta aaacagcttt 76680 atagaaagtg gttgatgttc tcagcaaaac attgtctcca tacctttaat tttaaaaatt 76740 ctccattttc atcatttcag ttatactcag atacaaataa ctttcaaatt tatattaata 76800 tttttagccc atatatctcc ccatacttct aattcatatg tgcagctgat accaggatgt 76860 tgacccctga atcctatcca aaactatttt ctccctagca agtttcttct cctttatttt 76920 atctcagcaa tgctgttatc atcatctacc cttcaagaca aatactgaaa gatgtatttg 76980 attccttctt cttcccatct accggcaatc agttgacaaa tgtcttttat ttttcccttt 77040 aaatattttt ctgatgcact ctctcctctt aattccaatg tgttgacttg acttacttca 77100 ggcttttatc atctctcacc tgggctttga cagtatctcc caactgtcct ccctaactcc 77160 agacttattc ccttgaatcc acctaccaca tagctaagaa tgatccatct acaatgcaag 77220 gctatgtcac tcccattgct ccttaataga cttgcaagac cttggatgat ctgacccagc 77280 ctacatctct aaacacatct ttgcttctcc cacctttaag ttactcattg ttgaactggt 77340 gatcttccct tgagaattgg gaagacatgg agagacttgt gatgattaag tcaagattca 77400 tttgcccttt ccctgccatc cttctgccac caacaccatg gaacagccta aaagaggatg 77460 aaagaccaaa gcattcacac catacccccc aatacacaca cacacacaca cacacacaca 77520 cacacacaca cacacacaca cacagattct aaatctctta agccacacgg aagtttaaaa 77580 caagactgga ttaagtttca gagaacgaaa gtgacctgat gactgtggaa tctacccgac 77640 atagttaagg gttgttaaaa gaaaattaga tataacacaa ctgaaagcag tggttttaca 77700 aagtaagacc agtttatgtt taaatcccaa agaactgaga tttaacgaac tgattatatt 77760 ccattcaggc attatctctc tttttttcta gaaagttttt cctgacctcc ctcccgattg 77820 actgatacac tcccttttaa ccttctctac cccagcactc atgtgcttgt ttctcctgtt 77880 gagttgtgag ctgcttttga ggctaggatt gactcttact catcttcatt tttatacagt 77940 gcctggcacc caaatggttc ttattaaatg ggtgttgcat gaatgagtga tggatgatgg 78000 attcacttag caaaatccca gaaatgctaa atcacagtga ctaatttcaa gtgacttcca 78060 aagttgctga tattttaggc tgtattattt attaatgttt tctgattttc agtgtaaaat 78120 atgttgggat tgaaaacatt ttattaccct acgcatacct attttcagat ttatatgtat 78180 tttcaacatt tttgagataa ttcagtcatt tttatggtca ctgtattttt aaatttgttt 78240 tgtaggtttt cgaaacttac atattgatga ccagataact ctcattcagt attcttggat 78300 gagcttaatg gtgtttggtc taggatggag atcctacaaa cacgtcagtg ggcagatgct 78360 gtattttgca cctgatctaa tactaaatga gtaagtagta acttttgttg tttttgttat 78420 tttaagtgta catgtaggat aattttgaaa gttatatttc aatagatgat cacatattta 78480 gtgttcttga tatgatgaca ttactgtcat tttacggtaa ttttatattt ggagcttttt 78540 cctcttgttt caaaatatag ttatatcaaa tccaattttc ttacacatca agaatgtatc 78600 aagaaatata tttaaagata aacttaaatg tttcattaat caatttaaaa tttcatagct 78660 tggtactaat gacaataata aatgagaaat atttttgtta ctgacttaca ttcaaaaagt 78720 aaaaacatct ttcttcatac atgacaaatt agactgctaa tttatttaaa aaattgaaag 78780 ataaatattt caatttgctt tatatttaaa taattttaat taattttcta cctacaacct 78840 ttaactgaat aagaatcttt tcatggtaat atttatattg ttctagtggt aaatgctgac 78900 ttagaagtat ataactcaca cttatataag tctttacagt ttttagataa ttttcatgtt 78960 tcattacatt ttatccaaca attccttgag ataagtaagg aatcagcatt cttcattgta 79020 cagtttagaa aactgagtct caggagaatt atataatctg ccaaataatt tgaatgacag 79080 tgagattcct tatctctttt tagccaaaat ctagttttca ttattcttgt tagcttcatt 79140 tgactaatgt ggacattata aaacaacaca aggttgggaa aatgccttaa ttaaattttc 79200 tcagccttca gcacttgtgt ttaactttgt caattaatta atgcttgcca atgtcctctt 79260 ttactctttt ctgtatcatt gtatatttcc ctagaaaagt ttaaaatatg gtaagctttg 79320 tgaactgact tctcctattt aattgcacac aaaattatat aatttttcat agaaaataaa 79380 ttgtaagtaa ataaaacatt tctgagcatt cttcagcttc acattctcaa ctcctacacc 79440 tctgctgtgt tttcttggtt taggtattta aagtgtgata ataaaagtga gaaacaatgt 79500 taatcttggt cactgtgtat ttaatataat tttctagttt taagatttct taacctcaaa 79560 aattcagtat tgtgaaaagt ttagaagatg tttaaaattc caaatattgg gtttgtttat 79620 taaaagtagt agtcattttc aataatatgt aaggtaatta ccaaaaccat attccctgat 79680 aaattaccta aactggctga gacactattt tttttcctgt tgctttttct tttgtgtatt 79740 gtgtgtgatg caagacagcg gatgaaagaa tcatcattct attcattatg ccttaccatg 79800 tggcagatcc cacaggagtt tgtcaagctt caagttagcc aagaagagtt cctctgtatg 79860 aaagtattgt tacttcttaa tacaagtgag tgagttcaag taacttaatg caagatatct 79920 agtttcttaa ttcattagaa aagttgcaaa caatatgatt atatagttat gtatgaggta 79980 gacgtcttgg attataagta taaagaagaa atacccaata tattgttata gacattaata 80040 aaattactgg attttttcat cttttaccta ccatataata ctaaaatagc ctcatcaatt 80100 tctttttatt ttagataaaa tgatttaatt actctttcat attcacctaa ttcagtaata 80160 taaactacag tcatgtgcca cataataata ttttggtcaa caacagacca catatacaat 80220 ggttccctaa gatgatagtg gagctgaaaa attatcatcg cctagtcctg ttatagccac 80280 cataacatca tagcacaatg cactaatcac atgttcgtgg tgatactaat gtaaacaaac 80340 ctactgtgct gccagtcata taaagtatag cacatacaat tatgtacagt acatagtact 80400 tggtaaagat aataaaaaac tcttactggt ttatgtattt actgtagtat acttttctat 80460 tgagagtcac tacttctact ttttttaagt taaccgcaaa acagcctcag gcaggtctgt 80520 cagagggatt ccagaaaagg gcatacgtca ccctgggaga tgacaactcc gtgtgtgtta 80580 ctgccccctg aagaccttcc agtgagataa gatgttgggg tggatgacag tgatattgat 80640 gattctgact ctgtgtaggt ctaggctaat gtgtgttttg ggtattagtt tttttaaaaa 80700 aagtttaagc aaaaaagaaa atatttaaaa atagataaag tttatagaat aaagatatag 80760 agaaagaaag ccaggcacgg tggcatgtgc ctgtagtccc cactacttgg gaggctgagg 80820 tgagaggatc acttgagccc aggagtttta agtccagcct aagcaacatg tgagaacttg 80880 tctctaaaaa aagaaagaaa gagaactgta ccttgtgttt ttgtttttaa ctaaatgtta 80940 ttacaaaaga gtcaaaaagt taaaaaacta gagtttataa agtaaaaaag ttacggtaag 81000 gtaaagctaa tttattataa aagaaataaa aatattttta taaatgtagt gtagcctaag 81060 tgtacagtgt ttataaaggc tatcgtagtg tatagtaatg tcctaggcct tcacattctg 81120 tcaccactca ctcgctgact cacgcagagc aacttccagt cctgcaagtt ccattcatgt 81180 taagtcccct atgcaagtgt cccattcgta tcttttatat tggattttta ctatgcctct 81240 tctgtgttta gatacaaaac acttaccatt gtgttacagc tgcctacagt attcagtaat 81300 cacatgctgt acaggttcat agcctaggaa taaggcctag gtgtatagta gtctatacca 81360 tctaggtttg tgtgagtaca ctctgtgata atcacacagt gatgaaatca cataaggaca 81420 catttctcag aatatatccc catcattaag tgatacacga ctgcaattaa tattggataa 81480 gcacaaacca tactctcctc taccaccact ctcaccccat ccccaaagta tttactatat 81540 aaaatacata attttgttta gaattatttt tatcaagata aattgttaaa acatgtaact 81600 ataaagatta gcatcttaat ataatagtgg tttctttatc actgacacag tccagtgagt 81660 tagcaagaaa cacccaggct ccttctagcc agtggcttca ccccttccta gtggcttgca 81720 aacctctatt ggacatgact agaaatgagc actgagtgag agagattttg aaggctagat 81780 ctgaaactat catacatcac tttcacccac attccaaggc acttaagctg caggaaaagc 81840 tgggaaatac agtctagcta taggcccaga agaaagagga aattatgttt tataaagcat 81900 tgcattgatc accagaagga caggcaaata tccgttccac tttttcttat atccaaggaa 81960 catacttacc tctgcctcag tggatcccca agggtctggg tgggcaaagt tttaaaaaaa 82020 agaaaaagca gctgctttga cccttgcata ggcaggatca gccctcaaat gtacaaactg 82080 atgcttttgt gattcattgt gatttctatg acaaccacag ggagctaaga cctcaggccg 82140 agggtgagcc taaggtcttt cttggcaaca tctttacagg gacttctttc taaggtccca 82200 aatctcaaat ggctaggggg tgcagatcag gagggaaggg caggagagat aaaccctgaa 82260 atggaaggct tcctcataga atttatgcaa agacagggat gaatacataa ccatcttggg 82320 aaccctattt ggacaaggaa tttcaaggct aatttggtga cagagcatca attgaacaaa 82380 ggatatttac gtggaaccgg cccagagagt caactggaat acatttaata atacagtaat 82440 tagctcctgc ccttctttgc atgaaggaat gaatcctata gcacttatgt cacagatagt 82500 cctagatctt ccaaactggc ttatatcctt aacataattc tctatttatg gataggcaac 82560 attatgctgg ggaattaaac aacaatggaa ttaatcaaca gggaggcttt gctcattgtt 82620 gtcacttaga aacttaggcg atcactagag cagtggtaag aaaaggaagt gagattcata 82680 caacttccac tcacatacca tagagagtaa gatttttagc catttgccac ttcacaaaaa 82740 ggaagaaaaa gaaactattt aatgaatagt acttacgctt tccacaaatt ctaatcaaga 82800 ttctaatgag atttggggag aagagggtga acttgacaaa cctactgaaa atccatcttt 82860 aaaactagaa gaataaatag ataagcatta taaaaatatg cagggaatta gaaagctgct 82920 gttaattaat acagcatggg agaagagata tactgaactt ttgggcaaat cagacaagta 82980 tatattagaa tataaatagt atagtggaaa ccacaaatca atgagtctga aacaaataaa 83040 tgttaagaaa aatagtttta tatttggaaa aaaactgttt ggatatgtag ctcaaattat 83100 atccttgaat aacagataat tgagttaaat gtaaacactc aaataattaa aaaacaagct 83160 agaagaaata gaaataatct tcaatctcaa atacctttta taaaataaat gcaaggagaa 83220 gattggcaaa gtaaaacttt gataatttgg tagttatgaa cttaaacctc tcatttgcca 83280 ttaaatatga aaatataaat ctccaggaat agacagtagg ttgaacaaac atttttacat 83340 tgttctctcc aaaattacac taaaggccag atgtagtcgg tcacatttct aatcacactt 83400 tgggagggca agctgggcag atggcttgaa cccaggagtt tgagaccagc ctaggcaact 83460 tggaaaaccc cgtctctaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 83520 attagccagg cgtagtggca agtgcctgta gtcccaggta gccgggaaac tgaggtggga 83580 ggatcacctc aacctaggaa gtcgaggctg cagtgagcca tgattgtgcc actgtactcc 83640 tgcctgggtg attgagtgag accatgtctc aaaataaaaa taaataaata aataaataaa 83700 aataaataaa ttacactaaa atcttagtaa agggatttac tatgaaaaat taagaaaaaa 83760 tgattttaca ttcatgttta cagctgaggg aaatataggg attggttggt aaccttaaag 83820 agagctgtga aagtttgaaa tgactgctat ttgaagtagt ccagaaaatt agtacagcct 83880 ctatggaaca ccatatggag atttctcaaa aaactataaa tataactacc attctatcta 83940 gcaatcccac cactggatat ctacccaaag gaaaagaaat cattatgtca aaaagacacc 84000 tgcactcttg tttattacag cagtattcac tatagcaaag ataggaaatc aatccaagtg 84060 tgtatcaaca gataatagga taaagaaaat gtgatgtact cacacatata catacataca 84120 cacacacaca cacacacaca cacacacaaa atggaatagt attcagcaat caaaaagaat 84180 gaaagtgtgt cttttgcagc aacatggatg gaacaggaag tcattatctt aagtgaaaca 84240 attcagagac aaagtcagat accacatgtt ctcgcttata agtgggagct aaataatgca 84300 tgtacatggg catagagtat agaataattg acagtggtgc cttagaaggc tggaaaggta 84360 ggaggggggt gagacattac ctaatggata caatgtgcat tattgggtga tggttatacc 84420 aaaagctcag atttcaccac tatgcaatat attcatgtaa caaaactgca cagatggtca 84480 ttaaatttat acaaatttaa taaaaagagg ttctagatat acatatagat gtacatacta 84540 gagttaagtt ttcaattgac aaaaatttgc atagatatac tgaattgcca gaattactct 84600 tcagagtctc ctttatatac aagatcttga aaactcaaaa ccaaagaatt aacttatttg 84660 tttttcataa gtttgtatct ctggccttct tttctagggg aaatggaaac caagttttga 84720 ttggggtgca ggggataaat acaggcttaa gaaatagtaa ttttcctccc tgttccctga 84780 gatacaacaa tattggaatt aggccaaggc tgggcatggt ggctcaagcc cgtaattcca 84840 gcactttggg aagctgaggt gggcgaatca cttgaggtga ggagttcaag accagcctgg 84900 ccaatatagt gaaaccccat ctctaccaaa aagacaaaaa ttagtcggac atgatggcac 84960 acacttgtaa tcctagctac tcaggaggct gaggcgggag aattgcttga acatgggagg 85020 cggaggttgc agtgacctga gattgtgcca ctgaactcca gcctgggcga cagagcaaga 85080 ccctgtgtca aaaaaaaaaa aaaaaaaaaa aaagcaatta ggccaactaa caatcttgga 85140 atggcctctc agtgttccag taaaagaaag agttgcaagt ctgaggtaga aatgattaag 85200 cttaattagg aaggcatgtc aaaagttgat ataggttgaa aactaggtct cctgagccaa 85260 gcaactagcc aagtttcaaa tacaaagtaa gagttcttga aggactttaa aagtgccatt 85320 ccagtgaaca catgaatgat aagcaaaacc gccttattgc tgatatggag aaagttttag 85380 tggtctgaat agaagatcaa accagccaca atattccctt aagcctaagc ctcatccaga 85440 ggaaggccct gattctcctc aatactagga aggcaaagag atgtaaggaa ggagcagaaa 85500 aaagtttgaa gctagcagaa gttggttcat gagatttaag aagccatttc cataacaaag 85560 tataaggtga agcagcaagt gctgatgcag aagctgcagc aagttaccca gaagatctag 85620 gtaagattat cgctacttat gaaagtagct gtgctaaaaa acaggttttc agggtagata 85680 aaacagcttt ccactggaag aagatgtcat ctaggacttt catagctaca gagaacaagt 85740 caatgtctgt cttcaaagct tcaaatgaca ggttgactct cttgttaggg gctaatgaag 85800 ctggtgactt gaagctgaag ctggtgactt gaagctgaag ctaatgctca tttatcattc 85860 tgaagatcct aaggccttta agaattacgc tcaatctgtt ctgcctgtgg tttatgaatg 85920 gaacaacaaa gctggatgac agcacatcta tttatggcat ggtttactga atattttgac 85980 ccactattga gacttactgc tcagaaaaga atactacttt caaatattat tgctcattga 86040 caatgcacct agtcagccaa gagctcttat ggatatatac aaggagatta atgttttcat 86100 ttctgctaac acaacattca ttctgtagtt catagatcaa agagtaattt tccactttca 86160 tgtcttatta tttaagaaat atattttgta aagctgtggc tgccatagat agtgtttcct 86220 ctgatggatc tgggtaaagt aaattgaaaa ccttctagaa aggattcacc tttctagatg 86280 ccattaagaa cattcgtgat tcatgggaga aggtgaaaat agcaacatta acaagagttt 86340 ggaagaagtt tatttcatcc ctcttgcatg actttgaggg tgttcaaaac ttcagtggag 86400 gaaataacta cagatatgtg aaaagagcaa gagaactaaa attagaagta aagcctgaag 86460 acatgactga attgaagagg tttcgtggta aaacttgaaa gatgaggagt tgtttcttat 86520 ggatgagcaa agaaagtggt ttcttgagtt ggaatctact tctagtgaag atgctatgaa 86580 gattattgaa gtgacaacaa aggatttcgg agattacata aacttagttg ataatgcagc 86640 agcaggactt aagaggattg actccaatta tgaaagaagt tctattgtgg gtaaattgcc 86700 atcaaactgc tagcattgca tgctacagaa aaattattca caaaagagtc aactgatgca 86760 gcaaacttca ttgttgcctt attttgagaa actgtcatag ccaccccaac cttcagcaac 86820 caccaccctg gccaatcagc agccatcaac atttagacaa caccctccaa cagcaaaaag 86880 attttgactc agggaagact cagatgattg ttaccatttt ctagcaataa agtgtttttt 86940 agttaaggtg tgttcacttt ttaaataatg ctgttgcaca cttaatacat tctagtatag 87000 tgtaaatata acttctacgt ggactgggaa acaaaaagat gcatgtgatt cactttgttg 87060 caatatttcc ttcattgccg tggtctggaa ccaaaccggc tatatctcca agatatgcct 87120 gtgtaacaaa gtggctggat ataagataaa tatttaaata ttaatacctt tctcccatat 87180 cagcaggaag gcattagaaa acttaatgaa gcaattttta aaattacaac aaaaagataa 87240 atcatctagg aataatctta atggcaaata cttgagattt atataaagaa gacaaaagtt 87300 tactaagaga tccaagtatt cattaaaaag aagggaaaaa aaagatccag attacatgca 87360 gctgtaaact aagtcaaacc aagtatttaa gaagcatgta gaataaggcc aggttctctt 87420 ttgagcaaat gctaagttaa atgcacaatc cattgataca ggtctaaaat aggtcagctg 87480 aagtgttctt taatatacct attaattctt atgttcttcc agtgaactag aagtttatat 87540 gtcctgatat tgatagtgat atcaataaca ctatctcttt cctatataag gtgttttttt 87600 tctagagtca tgtatcagaa gaaatcatta tatagctcag aaatatatgt tataaattgt 87660 taatgtcatc tttttataca caactgccac ttttaattgt cttcttaaca gttcctttgg 87720 aagggctacg aagtcaaacc cagtttgagg agatgaggtc aagctacatt agagagctca 87780 tcaaggcaat tggtttgagg caaaaaggag ttgtgtcgag ctcacagcgt ttctatcaac 87840 ttacaaaact tcttgataac ttgcatgatg taagtatttg gttgattcca gaatatcaat 87900 gattattctc tgaatttcta taacttttta aatgttacat gtaaatttta ctttgtatga 87960 ttttctcaga ttaatacttg tatgttaaaa gtgtttggat catgctgctc agacttttta 88020 ttgtgttttt ttttcatttt caatatagac cactcaaata ttcttttaca agtatttgta 88080 taatgtgagg gtaatttaat agctgataga aattatggta catttcctga atatgttcta 88140 aaatatttgt gcctaaaatt ataaaagatt ttatatatac agtaaaaaca aaggtatgta 88200 tgtgaaaaat aagactcatg ccattttcag tggaattata aataacattg ctgatgggtg 88260 ggcaatcagg caatacatag caaaagttaa aatttgcatg cttttcaagt ttctattttt 88320 taggatttat cttaaagaaa taattagaca aatgtaatat acatgcaagg atgttcacca 88380 tagcagaggg ggaaaaaaga aaagcaaaca agaaatgccc aacaccagag gtggattact 88440 aagacaagat atctgtggtt tgtcaagata tatgaaatac aagcttttat tgctactatg 88500 ttcagatcac attaaaatga cagtgaaggc ataaaaaagc tatccactct ccaggtcaac 88560 ttttgaggtc ttactcctga atacgaacag acagccaaga ttatgagcta tttgagtaaa 88620 gcctctaaaa tgaaaggcag aaagcaaaat aaaaagaaac ttgaaggaaa taaacaccag 88680 tgcagagaac agaagaaaac ttctaacatc ctcagagaaa taagaatgat acggtatcca 88740 tgacatgaga acagaaaaca tttttaaaac agacatttag caagaaaata cttttggata 88800 attaaatgaa gaaaggcaga aattgaaaat tcaatgaaag ggctgaaatg gaaaggaaaa 88860 tctgaggaaa aataaaagat caaaacatgg aaaattagaa agaagagtta tagtaaattt 88920 agtattcagg agattctagc ctccaattaa taggaatgtc cagaagaaaa aatagagaaa 88980 atcaaggaga gaaatttgtg taagaaatcg caaaagttgc tggaactgaa acatacaagt 89040 tttctaacaa aaagggtccg gtgtagaggc actgcacaat tgaactaagg tacattattg 89100 taaaattgta ccaggccaga aatgaagaga atattttaaa gtataccagt gatgaaagaa 89160 acaggtcaca tagacgttat caggattcag aatgatgttg gacttctctg cagcaacact 89220 ggaagccaga agacagtgga gcagtgcctt cagaatttga tggaaaagaa aatcttcaaa 89280 gaatttcata cctagccaaa ccatcaaaca agtacaatgg cagcatgaaa gtattttcca 89340 acttgcaagg tctcctagag tttacctcag atacaacttt ctcaagaact gctacaggat 89400 gtcttataac aaaacatggg attaagccaa agatccaccc caacagagat gcaaagtgat 89460 gtcccagaac aataactgtg cagagcagta gttagtctag atgggagcag taatacaaag 89520 ggttctagga taccagctcc aggaaaaata aatggaactg agagattacc actaagtatg 89580 actgtaattg aggtgatttt tacaattctg ttgcagaatt tgactatggc tttgagacaa 89640 atatagagaa aacaaagcaa acaagaacac gaggtgattc acttttgcaa aataaaaata 89700 agtcgtaaga atggagatgc aatccagcag aaaaccataa aaaaatcttt atgatataaa 89760 tgctggatat tgactttagc taaaaattga gccaaaagct ctctctgggg agaagagaac 89820 atgtatgtag ttgaggaaga gagtgtctag tataagagag cttcattttc ttcatttcct 89880 atagtagaaa ctaaaactaa ctttagtgga aatatagaaa tatcaaaaga cacaggctac 89940 aaaaagttga aggtagttgt gtctagggag ctggaatagg gatgggaaga agtctatcag 90000 gaaactgcta ggctctattg atgctgctgc tgctgctgtt tttgttttta taatcatgtg 90060 gtactacttg actttcaaca ttatacacat gaattacttt gataaaaatt aaacgatgtt 90120 ggattattgg cataggaaga accaatatgt tgttatataa aacagctaga ttagcaaata 90180 gaatttatga aacatgtgta atattgtgtg tatgcttggg tatgtctgta tacatgtata 90240 aaaagaaaaa aaaacttgag taatacacca aaatgttcac acagcttatc ctgtagaatt 90300 gtgaataact ttcatttctt taaataaatc tttccataat atcagaatgt tctatcatga 90360 gtatataaca ttttacatat tattctagaa aatggtggga gttattttca ctatggtaag 90420 aaaaaaacac cttgagaagt atttatatta taaatagtta gaaaaataaa ttgccatgtt 90480 tgaatagcat atgaatttat tatttttatt acatgttttc tactcatttg ttaaaccaac 90540 agcttgtcaa acaacttcat ctgtactgct tgaatacatt tatccagtcc cgggcactga 90600 gtgttgaatt tccagaaatg atgtctgaag ttattgctgc acaattaccc aagatattgg 90660 cagggatggt gaaacccctt ctctttcata aaaagtgaat gtcatctttt tcttttaaag 90720 aattaaattt tgtggtatgt ctttttgttt tggtcaggat tatgaggtct tgagttttta 90780 taatgttctt ctgaaagcct tacatttata acatcatagt gtgtaaattt aaaagaaaaa 90840 ttgtgaggtt ctaattattt tcttttataa agtataatta gaatgtttaa ctgttttgtt 90900 tacccatatt ttcttgaaga atttacaaga ttgaaaaagt actaaaattg ttaaagtaaa 90960 ctatcttatc catattattt cataccatgt aggtgaggat ttttaacttt tgcatctaac 91020 aaatcatcga cttaagagaa aaaatcttac atgtaataac acaaagctat tatatgttat 91080 ttctaggtaa ctccctttgt gtcaattata tttccaaaaa tgaaccttta aaatggtatg 91140 caaaattttg tctatatata tttgtgtgag gaggaaattc ataactttcc tcagattttc 91200 aaaagtattt ttaatgcaaa aaatgtagaa agagtttaaa accactaaaa tagattgatg 91260 ttcttcaaac taggcaaaac aactcatatg ttaagaccat tttccagatt ggaaacacaa 91320 atctcttagg aagttaataa gtagattcat atcattatgc aaatagtatt gtgggttttg 91380 taggttttta aaataacctt ttttggggag agaattgtcc tctaatgagg tattgcgagt 91440 ggacataaga aatcagaaga ttatggccta actgtactcc ttaccaactg tggcatgctg 91500 aaagttagtc actcttactg attctcaatt ctctcacctt tgaaagtagt aaaatatctt 91560 tcctgccaat tgctcctttg ggtcagagct tattaacatc ttttcaaatc aaaggaaaga 91620 agaaagggag aggaggagga gggaggtatc aattcacata cctttctcct ctttatcctc 91680 cactatcatg aattcatatt atgtttcagc catgcaaatc tttttaccat gaaatttctt 91740 ccagaatttt ccccctttga cacaaattcc atgcatgttt caaccttcga gactcagcca 91800 aatgtcattt ctgtaaaatc ttccctgagt cttccaagca gtaatttgcc ttctcctaga 91860 gtttacctgc cattttgtgc acatttgagt tacagtagca tgttatttta caattgtgac 91920 tctcctggga gtctgggagc catataaagt ggtcaatagt gtttgctgac tgagagttga 91980 atgacatttt ctctctgtct tggtattact gtagatttcg atcattcttt ggttacattt 92040 ctgcatattt ctgtacccat gactttatca ctttcttctc ccatgcttta tctccatcaa 92100 ttatcttcat tacttttaaa ttttccacct ttgcttccta ctttgtgaga tctctccctt 92160 tactgactat aacatagaag aatagaagtg tattttatgt gtcttaagga caatacttta 92220 gattccttgt tctaagtttt taaactgaat gaatggaata ttatttctct ccctaagcaa 92280 aattccacaa aacaattatt tcttatgttt atgtagcctt aaattgtttt gtactgtaaa 92340 cctcagcata aaaactttct tcatttctaa tttcattcaa caaatattga ttgaatacct 92400 ggtattagca caagaaaaat gtgctaataa gccttatgag aatttggagc tgaagaaaga 92460 catataactc aggaaagtta cagtccagta gtaggtataa attacagtgc ctgataaata 92520 ggcattttaa tatttgtaca ctcaacgtat actaggtagg tgcaaaacat ttacatataa 92580 ttttactgat acccatgcag cacaaaggta ctaactttaa atattaaata acacctttat 92640 gtgtcagtaa ttcatttgca ttaaatctta ttgaaaaggc tttcaatata ttttccccac 92700 aaatgtcatc ccaagaaaaa agtattttta acatctccca aatataatag ttacaggaaa 92760 tctacctctg tgagagtgac acctctcaga atgaactgtg tgacacaaga aaatgaatgt 92820 aggtctatcc aaaaaaaacc ccaagaaaca aaaacaatat tattagccct ttatgcttaa 92880 gtgatggact cagggaacag ttgatgttgt gatcatttta ttatctgatt cttgttactt 92940 tgaattaaac caatattttg atgatataaa tcatttccac cagcatatat ttaatttcca 93000 taataacttt aaaattttct aatttcactc aactatgagg gaatagaatg tggtggccac 93060 aggtttggct tttgttaaaa tgtttgatat cttcgatgtt gatctctgtc tgcaatgtag 93120 atgtctaaac actaggattt aatatttaag gctaagcttt aaaaataaag taccttttta 93180 aaaagaatat ggcttcacca aatggaaaat acctaatttc taaatctttt tctctacaaa 93240 gtcctatcta ctaatgtctc cattactatt tagtcatcat aaccattatc ttcattttac 93300 atgtcgtgtt ctttctggta gctctaaaat gacactaaat cataagaaga caggttacat 93360 atcaggaaat acttgaaggt tactgaaata gattcttgag ttaatgaaaa tattttctgt 93420 aaaaaggttt gaaaagccat ttgagtctaa agcattatac ctccattatc agtagttatg 93480 tgacaattgt gtgtgtgttt aatgtttaaa gatgtggcac tttttaataa ggcaatgcta 93540 tgctattttt tcccatttaa cattaagata atttattgct atacagatga tatggaaata 93600 tgatgaacaa tatttttttt gccaaaacta tgccttgtaa gtagccatgg aatgtcaacc 93660 tgtaacttaa attatccaca gatagtcatg tgtttgatga tgggcactgt ggagataact 93720 gacataggac tgtgcccccc ttctctgcca cttactagct ggatgagatt aagcaagtca 93780 tttaactgct ctgattaaac ctgcctttcc caagtgcttt gtaatgaata gaaatggaaa 93840 ccaaaaaaaa cgtatacagg ccttcagaaa tagtaattgc tactattttg ttttcattaa 93900 gccatagttc tggctataat tttatcaaac tcaccagcta tattctacag tgaaagcagg 93960 attctagaaa gtctcactgt tttatttatg tcaccatgtg ctatgatata tttggttgaa 94020 ttcatttgaa attagggctg gaagtattca agtaatttct tctgctgaaa aaatacagtg 94080 ttttgagttt agggcctgtt ttatcaaagt tctaaagagc ctatcactct tccattgtag 94140 acattttaaa ataatgacac tgattttaac atttttaagt gtctttttag aacagagagc 94200 ctgactagaa cacagcccct ccaaaaaccc atgctcaaat tatttttact atggcagcaa 94260 ttccacaaaa gggaacaatg ggtttagaaa ttacaatgaa gtcatcaacc caaaaaacat 94320 ccctatccct aagaaggtta tgatataaaa tgcccacaag aaatctatgt ctgctttaat 94380 ctgtctttta ttgctttgga aggatggcta ttacattttt agtttttgct gtgaatacct 94440 gagcagtttc tctcatccat acttatcctt cacacatcag aagtcaggat agaatatgaa 94500 tcattttaaa aacttttaca actccagagc catgtgcata agaagcattc aaaacttgcc 94560 aaaacataca ttttttttca aatttaaaga tactctattt ttgtattcaa tagctcaaca 94620 actgtggtcc ccactgataa agtgaagtgg acaaggagac aagtaatggc ataagtttgt 94680 ttttcccaaa gtatgcctgt tcaatagcca ttggatgtgg gaaatttcta catctcttaa 94740 aattttacag aaaatacata gccagatagt ctagcaaaag ttcaccaagt cctaaattgc 94800 ttatccttac ttcactaagt catgaaatca ttttaatgaa aagaacatca cctaggtttt 94860 gtggtttctt tttttcttat tcatggctga gtgaaaacaa caatctctgt ttctccctag 94920 catctgtgga ctatttaatg taccattatt ccacactcta tggtccttac taaatacaaa 94980 attgaacaaa aagcagtaaa acaactgact cttcacccat attataaaat ataatccaag 95040 ccagattagt caacatccat aagatgaatc caagctgaac tgggcctaga ttattgagtt 95100 caggttggat cacatcccta tttattaata aacttaggaa agaaggcctt acagaccatc 95160 agttagctgg agctaataga acctacactt ctaaagttcg gcctagaatc aatgtggcct 95220 taaaagctga aaagaagcag gaaagaacag ttttcttcaa taatttgtcc accctgtcac 95280 tggagaaaat ttaagaattt gggggtgttg gtagtaagtt aaacacagca gctgttcatg 95340 gcagaaatta ttcaatacat accttctctg aatatcctat aaccaaagca aagaaaaaca 95400 ccaaggggtt tgttctcctc cttggagttg acctcattcc aaggcagagc tcaggtcaca 95460 ggcacagggg ctgcgcccaa gcttgtccgc agccttatgc agctgtggag tctggaagac 95520 tgttgcagga ctgctggcct agtcccagaa tgtcagcctc attttcgatt tactggctct 95580 tgttgctgta tgtcatgctg accttattgt taaacacagg tttgtttgct ttttttccac 95640 tcatggagac atgggagagg cattattttt aagctggttg aaagctttaa ccgataaagc 95700 atttttagag aaatgtgaat caggcagcta agaaagcata ctctgtccat tacggtaaag 95760 aaaatgcaca gattattaac tctgcagtgt ggcattagtg tcctggtcaa tattcggata 95820 gatatgaata aaatatttaa atggtattgt aaatagtttt caggacatat gctatagctt 95880 atttttatta tcttttgaaa ttgctcttaa tacatcaaat cctgatgtat tcaatttatc 95940 agatataaat tattctaaat gaagcccagt taaatgtttt tgtcttgtca gttatatgtt 96000 aagtttctga tctctttgtc tatgacgttt actaatctgc atttttactg ttatgaatta 96060 ttttagacag cagtggtttc aagctttttg ccactaaaaa taccttttat tttctcctcc 96120 cccagaaaag tctatacctt gaagtatcta tccaccaaac tgtacttcta ttaagaaata 96180 gttattgtgt tttcttaatg ttttgttatt caaagacata tcaatgaaag ctgctgagca 96240 gcatgaataa caattatatc cacacagatt tgatatattt tgtgcagcct taacttgata 96300 gtataaaatg tcattgcttt ttaaataata gttagtcaat ggacttctat catagctttc 96360 ctaaactagg ttaagatcca gagctttggg gtcataatat attacataca attaagttat 96420 ctttttctaa gggctttaaa attcatgaga ataaccaaaa aaggtatgtg gagagttaat 96480 acaaacatac catattcttg ttgaaacaga gatgtggctc tgcttgttct ccataaggta 96540 gaaatacttt ccagaatttg cctaaactag taagccctga atttgctatg attagggata 96600 ggaagagatt ttcacatggc agactttaga attcttcact ttagccagta aagtatctcc 96660 ttttgatctt agtattctgt gtattttaac ttttctgagt tgtgcatgtt tataagaaaa 96720 atcagcacaa agggtttaag ttaaagcctt tttactgaaa tttgaaagaa acagaagaaa 96780 atatcaaagt tctttgtatt ttgagaggat taaatatgat ttacaaaagt tacatggagg 96840 gctctctaaa acattaaatt aattattttt tgttgaaaag tcttacttta ggcatcattt 96900 tattcctcag caactagctg tgaagccttt actgtgctgt atgccagtca ctctgctaga 96960 ttgtggagat taccagtgtt cccgtcttct ccgagcttag agttggatgg ggaataaaga 97020 caggtaaaca gatagctaca atattgtact gtgaatgctt atgctggagg aagtacaggg 97080 aactattgga gcacctaaga ggagcaccta ccttgaattt aggggttagc agaggcatcc 97140 tgaaaaaagt caaagctaag ccacaatcta taagcagttt aggaattagc agaacgtgcg 97200 tggtgaggag atgccaaagg caagaagaga agagtattcc aaacaggagg gattccaaag 97260 agagaagagt atcccaaaca acatttgcac aaacctgatg gggagagaga atgtggggtg 97320 gggatggatg atgagactga agaagaaagc caggtctaga taatcagtgg ccttgtacac 97380 catgttaaag agtgtagact tgattctgtt gtaaacagga aagcagcaca attcatatga 97440 atattttaga agactcccac tggaatatgg agaataaagt tggagatgac taatcctgga 97500 agcagggaga acatttttga ggaagttgca ctattttggt gaaaatgatg atcataaaca 97560 tgaagaattg taggtgatca tgacctcctc tctaattttc cagaagggtt ttggaagata 97620 taacatagga acattgacag gactgacgaa aggagatgaa atacaccata taaattgtca 97680 aacacaaggc cagatgtcta attattttgc ttatgtgttg aaattacaaa tttttcatca 97740 ggaaaccaaa aactacaaaa cttagttttc ccaagtccca gaattctatc tgtccaaaca 97800 atctgtacca ctccacctat atccctacct ttgcatgtct gtccaacctc aaagtccagg 97860 tctatacaca cgggtaagac tagagcagtt caagtttcag aaaatgagaa agaggaactg 97920 agttgtgctg aacccataca aaataaacac attctttgta tagattcttg gaacctcgag 97980 aggaattcac ctaactcata ggtatttgat ggtatgaatc catggctggg ctcggctttt 98040 aaaaagcctt atctgggatt ccttctatgg aaccaagttc catcaaagcc catttaaaag 98100 cctacattaa aaacaaaatt cttgctgcat tgtatacaaa taatgatgtc atgatcaaat 98160 aatcagatgc cattatcaag tggaattaca aaatggtata cccactccaa aaaaaaaaaa 98220 aaagctaaat tctcagtaga acattgtgac ttcatgagcc ctccacagcc ttggagctga 98280 ggagggagca ctggtgagca gtaggttgaa gagaaaactt ggcgcttaat aatctatcca 98340 tgttttttca tctaaaagag ccttcttttt ggattacctt attcaatttc catcaaggaa 98400 attgttagtt ccactaacca gacagcagct gggaaggcag aagcttactg tatgtacatg 98460 gtagctgtgg gaaggaggtt tctttctcca ggtcctcact ggccatacac cagtcccttg 98520 ttagttatgc ctggtcatag acccccgttg ctatcatctc atatttaagt ctttggcttg 98580 tgaatttatc tattctttca gcttcagcac tgcagagtgc tgggactttg ctaacttcca 98640 tttcttgctg gcttagcaca ttcctcatag gcccagctct tttctca...

Examples

example 1

Cancer Patients Undergoing HER2 Targeted Therapy and Chemotherapy Benefit from PD-L1 Inhibitor Cotherapy, if the Expression Level of ER is Low or Absent (ER Negative) and if PD-L1 Expression Level is Increased

[0283]Estimation of gene expression was performed with the help of R Bioconductor package ‘affy’, R version 2.15.0. All exploratory analyses and predictive models were made using SAS JMP ver. 10.0

[0284]48 HER2+, ER+ and 39 HER2+, ER− breast cancer biopsies were obtained from NeoSphere clinical trial. The samples had been taken at diagnosis from patients afterwards treated with Docetaxel and Trastuzumab in a neo-adjuvant setting. The distribution of main clinical covariates at base line, as well as of clinical response (as assessed at the surgery) in the involved population is as follows:

ER Negative Samples:

Patient Age (see FIG. 17)Quantiles100.0%maximum72 99.5%72 97.5%71.55 90.0%64 75.0%quartile54 50.0%median50.5 25.0%quartile44.25 10.0%39 2.5%34.675 0.5%34 0.0%minimum34LevelCo...

CROSS-REFERENCE RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent application Ser. No. 17 / 744,271, filed on May 13, 2022 (Pending), which is a continuation of U.S. patent application Ser. No. 17 / 674,615 filed on Feb. 17, 2022 (Abandoned), which is a continuation of U.S. patent application Ser. No. 17 / 483,396 filed on Sep. 23, 2021 (Abandoned), which is a continuation of U.S. patent application Ser. No. 17 / 323,120 filed on May 18, 2021 (Abandoned), which is a continuation of U.S. patent application Ser. No. 16 / 814,688 filed on Mar. 10, 2020 (Abandoned), which is a continuation of U.S. patent application Ser. No. 15 / 815,384, filed Nov. 16, 2017 (Abandoned), which is a continuation of U.S. patent application Ser. No. 14 / 720,643, filed May 22, 2015 (Abandoned), which is a continuation of International Patent Application No. PCT / EP2013 / 075162, filed Nov. 29, 2013 (Expired), which claims priority to European Patent Application No. 12195182.6, filed Nov. 30, 2012 (Abandoned) and European Patent Application No. 12196177.5, filed Dec. 7, 2012 (Abandoned), the disclosures of each of which are incorporated by reference herein in their entireties.SEQUENCE LISTING

[0002] This application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Feb. 26, 2025 is named P31922-US-7_Sequence Listing.xml and is 625,775 bytes in size.BACKGROUND

[0003] The present invention relates to means and methods for determining whether a patient is in need of a PD-L1 inhibitor cotherapy. A patient is determined to be in need of the PD-L1 inhibitor cotherapy if a low or absent ER expression level and an expression level of programmed death ligand 1 (PD-L1) that is increased in comparison to a control is measured in vitro in a sample from the patient. The patient is undergoing therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway (like Trastuzumab) and a chemotherapeutic agent (like dodetaxel) or such a therapy is contemplated for the patient. Also provided herein are means and methods for treating a cancer in a cancer patient for whom therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway (like Trastuzumab) and a chemotherapeutic agent (like dodetaxel) is contemplated, wherein the patient is to receive PD-L1 inhibitor cotherapy.

[0004] The HER family of receptor tyrosine kinases are important mediators of cell growth, differentiation and survival. The receptor family includes four distinct members including epidermal growth factor receptor (EGFR, ErbB1, or HER1), HER2 (ErbB2 or p185neu), HER3 (ErbB3) and HER4 (ErbB4 or tyro2).

[0005] EGFR, encoded by the erbB1 gene, has been causally implicated in human malignancy. In particular, increased expression of EGFR has been observed in breast, bladder, lung, head, neck and stomach cancer as well as glioblastomas. Increased EGFR receptor expression is often associated with increased production of the EGFR ligand, transforming growth factor alpha (TGF-α), by the same tumor cells resulting in receptor activation by an autocrine stimulatory pathway. Baselga and Mendelsohn Pharmac. Ther. 64:127-154 (1994). Monoclonal antibodies directed against the EGFR or its ligands, TGF-α and EGF, have been evaluated as therapeutic agents in the treatment of such malignancies. See, e.g., Baselga and Mendelsohn., supra; Masui et al. Cancer Research 44:1002-1007 (1984); and Wu et al. J. Clin. Invest. 95:1897-1905 (1995).

[0006] The second member of the HER family, p185neu, was originally identified as the product of the transforming gene from neuroblastomas of chemically treated rats. The activated form of the neu proto-oncogene results from a point mutation (valine to glutamic acid) in the transmembrane region of the encoded protein. Amplification of the human homolog of neu is observed in breast and ovarian cancers and correlates with a poor prognosis (Slamon et al., Science, 235:177-182 (1987); Slamon et al., Science, 244:707-712 (1989); and U.S. Pat. No. 4,968,603). To date, no point mutation analogous to that in the neu proto-oncogene has been reported for human tumors. Overexpression of HER2 (frequently but not uniformly due to gene amplification) has also been observed in other carcinomas including carcinomas of the stomach, endometrium, salivary gland, lung, kidney, colon, thyroid, pancreas and bladder. See, among others, King et al., Science, 229:974 (1985); Yokota et al., Lancet: 1:765-767 (1986); Fukushige et al., Mol Cell Biol., 6:955-958 (1986); Guerin et al., Oncogene Res., 3:21-31 (1988); Cohen et al., Oncogene, 4:81-88 (1989); Yonemura et al., Cancer Res., 51:1034 (1991); Borst et al., Gynecol. Oncol., 38:364 (1990); Weiner et al., Cancer Res., 50:421-425 (1990); Kern et al., Cancer Res., 50:5184 (1990); Park et al., Cancer Res., 49:6605 (1989); Zhau et al., Mol. Carcinog., 3:254-257 (1990); Aasland et al. Br. J. Cancer 57:358-363 (1988); Williams et al. Pathobiology 59:46-52 (1991); and McCann et al., Cancer, 65:88-92 (1990). HER2 may be overexpressed in prostate cancer (Gu et al. Cancer Lett. 99:185-9 (1996); Ross et al. Hum. Pathol. 28:827-33 (1997); Ross et al. Cancer 79:2162-70 (1997); and Sadasivan et al. J. Urol. 150:126-31 (1993)).

[0007] Antibodies directed against the rat p185neu and human HER2 protein products have been described. Drebin and colleagues have raised antibodies against the rat neu gene product, p185neu. See, for example, Drebin et al., Cell 41:695-706 (1985); Myers et al., Meth. Enzym. 198:277-290 (1991); and WO94 / 22478. Drebin et al. Oncogene 2:273-277 (1988) report that mixtures of antibodies reactive with two distinct regions of p185neu result in synergistic anti-tumor effects on neu-transformed NIH-3T3 cells implanted into nude mice. See also U.S. Pat. No. 5,824,311 issued Oct. 20, 1998.

[0008] Hudziak et al., Mol. Cell. Biol. 9(3):1165-1172 (1989) describe the generation of a panel of HER2 antibodies which were characterized using the human breast tumor cell line SK-BR-3. Relative cell proliferation of the SK-BR-3 cells following exposure to the antibodies was determined by crystal violet staining of the monolayers after 72 hours. Using this assay, maximum inhibition was obtained with the antibody called 4D5 which inhibited cellular proliferation by 56%. Other antibodies in the panel reduced cellular proliferation to a lesser extent in this assay. The antibody 4D5 was further found to sensitize HER2-overexpressing breast tumor cell lines to the cytotoxic effects of TNF-α. See also U.S. Pat. No. 5,677,171 issued Oct. 14, 1997. The HER2 antibodies discussed in Hudziak et al. are further characterized in Fendly et al. Cancer Research 50:1550-1558 (1990); Kotts et al. In Vitro 26(3):59A (1990); Sarup et al. Growth Regulation 1:72-82 (1991); Shepard et al. J. Clin. Immunol. 11(3):117-127 (1991); Kumar et al. Mol. Cell. Biol. 11(2):979-986 (1991); Lewis et al. Cancer Immunol. Immunother. 37:255-263 (1993); Pietras et al. Oncogene 9:1829-1838 (1994); Vitetta et al. Cancer Research 54:5301-5309 (1994); Sliwkowski et al. J. Biol. Chem. 269(20):14661-14665 (1994); Scott et al. J. Biol. Chem. 266:14300-5 (1991); D'souza et al. Proc. Natl. Acad. Sci. 91:7202-7206 (1994); Lewis et al. Cancer Research 56:1457-1465 (1996); and Schaefer et al. Oncogene 15:1385-1394 (1997).

[0009] A recombinant humanized version of the murine HER2 antibody 4D5 (huMAb4D5-8, rhuMAb HER2, Trastuzumab or Herceptin™; U.S. Pat. No. 5,821,337) is clinically active in patients with HER2-overexpressing metastatic breast cancers that have received extensive prior anti-cancer therapy (Baselga et al., J. Clin. Oncol. 14:737-744 (1996)). Trastuzumab received marketing approval from the Food and Drug Administration Sep. 25, 1998 for the treatment of patients with metastatic breast cancer whose tumors overexpress the HER2 protein.

[0010] Humanized anti-ErbB2 antibodies include huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 (HERCEPTIN®) as described in Table 3 of U.S. Pat. No. 5,821,337 expressly incorporated herein by reference; humanized 520C9 (WO 93 / 21319) and humanized 2C4 antibodies as described in WO 01 / 000245 expressly incorporated herein by reference.

[0011] Pertuzumab (see e.g. WO 01 / 000245) is the first of a new class of agents known as HER dimerization inhibitors (HDIs). Pertuzumab binds to HER2 at its dimerization domain, thereby inhibiting its ability to form active dimer receptor complexes and thus blocking the downstream signal cascade that ultimately results in cell growth and division (see Franklin, M. C., Cancer Cell 5 (2004) 317-328). Pertuzumab is a fully humanized recombinant monoclonal antibody directed against the extracellular domain of HER2. Binding of Pertuzumab to the HER2 on human epithelial cells prevents HER2 from forming complexes with other members of the HER family (including EGFR, HER3, HER4) and probably also HER2 homodimerization. By blocking complex formation, Pertuzumab prevents the growth stimulatory effects and cell survival signals activated by ligands of HER1, HER3 and HER4 (e.g. EGF, TGFalpha, amphiregulin, and the heregulins). Another name for Pertuzumab is 2C4. Pertuzumab is a fully humanized recombinant monoclonal antibody based on the human IgG1(K) framework sequences. The structure of Pertuzumab consists of two heavy chains (449 residues) and two light chains (214 residues). Compared to Trastuzumab (Herceptin®), Pertuzumab has 12 amino acid differences in the light chain and 29 amino acid differences in the IgG1 heavy chain.

[0012] Other HER2 antibodies with various properties have been described in Tagliabue et al. Int. J. Cancer 47:933-937 (1991); McKenzie et al. Oncogene 4:543-548 (1989); Maier et al. Cancer Res. 51:5361-5369 (1991); Bacus et al. Molecular Carcinogenesis 3:350-362 (1990); Stancovski et al. PNAS (USA) 88:8691-8695 (1991); Bacus et al. Cancer Research 52:2580-2589 (1992); Xu et al. Int. J. Cancer 53:401-408 (1993); WO94 / 00136; Kasprzyk et al. Cancer Research 52:2771-2776 (1992); Hancock et al. Cancer Res. 51:4575-4580 (1991); Shawver et al. Cancer Res. 54:1367-1373 (1994); Arteaga et al. Cancer Res. 54:3758-3765 (1994); Harwerth et al. J. Biol. Chem. 267:15160-15167 (1992); U.S. Pat. No. 5,783,186; and Klapper et al. Oncogene 14:2099-2109 (1997).

[0013] Homology screening has resulted in the identification of two other HER receptor family members; HER3 (U.S. Pat. Nos. 5,183,884 and 5,480,968 as well as Kraus et al. PNAS (USA) 86:9193-9197 (1989)) and HER4 (EP Pat. Appln. No 599,274; Plowman et al., Proc. Natl. Acad. Sci. USA, 90:1746-1750 (1993); and Plowman et al., Nature, 366:473-475 (1993)). Both of these receptors display increased expression on at least some breast cancer cell lines.

[0014] The HER receptors are generally found in various combinations in cells and heterodimerization is thought to increase the diversity of cellular responses to a variety of HER ligands (Earp et al. Breast Cancer Research and Treatment 35: 115-132 (1995)). EGFR is bound by six different ligands; epidermal growth factor (EGF), transforming growth factor alpha (TGF-α), amphiregulin, heparin binding epidermal growth factor (HB-EGF), betacellulin and epiregulin (Groenen et al. Growth Factors 11:235-257 (1994)). A family of heregulin proteins resulting from alternative splicing of a single gene are ligands for HER3 and HER4. The heregulin family includes alpha, beta and gamma heregulins (Holmes et al., Science, 256:1205-1210 (1992); U.S. Pat. No. 5,641,869; and Schaefer et al. Oncogene 15:1385-1394 (1997)); neu differentiation factors (NDFs), glial growth factors (GGFs); acetylcholine receptor inducing activity (ARIA); and sensory and motor neuron derived factor (SMDF). For a review, see Groenen et al. Growth Factors 11:235-257 (1994); Lemke, G. Molec. &Cell. Neurosci. 7:247-262 (1996) and Lee et al. Pharm. Rev. 47:51-85 (1995). Recently three additional HER ligands were identified; neuregulin-2 (NRG-2) which is reported to bind either HER3 or HER4 (Chang et al. Nature 387 509-512 (1997); and Carraway et al Nature 387:512-516 (1997)); neuregulin-3 which binds HER4 (Zhang et al. PNAS (USA) 94(18):9562-7 (1997)); and neuregulin-4 which binds HER4 (Harari et al. Oncogene 18:2681-89 (1999)) HB-EGF, betacellulin and epiregulin also bind to HER4.

[0015] While EGF and TGFα do not bind HER2, EGF stimulates EGFR and HER2 to form a heterodimer, which activates EGFR and results in transphosphorylation of HER2 in the heterodimer. Dimerization and / or transphosphorylation appears to activate the HER2 tyrosine kinase. See Earp et al., supra. Likewise, when HER3 is co-expressed with HER2, an active signaling complex is formed and antibodies directed against HER2 are capable of disrupting this complex (Sliwkowski et al., J. Biol. Chem., 269(20):14661-14665 (1994)). Additionally, the affinity of HER3 for heregulin (HRG) is increased to a higher affinity state when co-expressed with HER2. See also, Levi et al., Journal of Neuroscience 15: 1329-1340 (1995); Morrissey et al., Proc. Natl. Acad. Sci. USA 92: 1431-1435 (1995); and Lewis et al., Cancer Res., 56:1457-1465 (1996) with respect to the HER2-HER3 protein complex. HER4, like HER3, forms an active signaling complex with HER2 (Carraway and Cantley, Cell 78:5-8 (1994)).

[0016] Also, antibody variant compositions are described in the art. U.S. Pat. No. 6,339,142 describes a HER2 antibody composition comprising a mixture of anti-HER2 antibody and one or more acidic variants thereof, wherein the amount of the acidic variant(s) is less than about 25%. Trastuzumab is the exemplified HER2 antibody. Reid et al. Poster presented at Well Characterized Biotech Pharmaceuticals conference (January, 2003) “Effects of Cell Culture Process Changes on Humanized Antibody Characteristics” describes an unnamed, humanized IgG1 antibody composition with N-terminal heterogeneities due to combinations of VHS signal peptide, N-terminal glutamine, and pyroglutamic acid on the heavy chain thereof. Harris et al. “The Ideal Chromatographic Antibody Characterization Method” talk presented at the IBC Antibody Production Conference (February, 2002) reports a VHS extension on the heavy chain of E25, a humanized anti-IgE antibody. Rouse et al. Poster presented at WCBP “Glycoprotein Characterization by High Resolution Mass Spectrometry and Its Application to Biopharmaceutical Development” (Jan. 6-9, 2004) describes a monoclonal antibody composition with N-terminal heterogeneity resulting from AHS or HS signal peptide residues on the light chain thereof. In a presentation at IBC Meeting (September, 2000) “Strategic Use of Comparability Studies and Assays for Well Characterized Biologicals,” Jill Porter discussed a late-eluting form of ZENAPAX™ with three extra amino acid residues on the heavy chain thereof. US2006 / 0018899 describes a composition comprising a main species pertuzumab antibody and an amino-terminal leader extension variant, as well as other variant forms of the pertuzumab antibody.

[0017] Patent publications related to HER antibodies include: U.S. Pat. Nos. 5,677,171, 5,720,937, 5,720,954, 5,725,856, 5,770,195, 5,772,997, 6,165,464, 6,387,371, 6,399,063, US2002 / 0192211A1, U.S. Pat. Nos. 6,015,567, 6,333,169, 4,968,603, 5,821,337, 6,054,297, 6,407,213, 6,719,971, 6,800,738, US2004 / 0236078A1, U.S. Pat. Nos. 5,648,237, 6,267,958, 6,685,940, 6,821,515, WO98 / 17797, U.S. Pat. Nos. 6,127,526, 6,333,398, 6,797,814, 6,339,142, 6,417,335, 6,489,447, WO99 / 31140, US2003 / 0147884A1, US2003 / 0170234A1, US2005 / 0002928A1, U.S. Pat. No. 6,573,043, US2003 / 0152987A1, WO99 / 48527, US2002 / 0141993A1, WO01 / 00245, US2003 / 0086924, US2004 / 0013667A1, WO00 / 69460, WO01 / 00238, WO01 / 15730, U.S. Pat. Nos. 6,627,196B1, 6,632,979B1, WO01 / 00244, US2002 / 0090662A1, WO01 / 89566, US2002 / 0064785, US2003 / 0134344, WO 04 / 24866, US2004 / 0082047, US2003 / 0175845A1, WO03 / 087131, US2003 / 0228663, WO2004 / 008099A2, US2004 / 0106161, WO2004 / 048525, US2004 / 0258685A1, U.S. Pat. Nos. 5,985,553, 5,747,261, 4,935,341, 5,401,638, 5,604,107, WO 87 / 07646, WO 89 / 10412, WO 91 / 05264, EP 412,116 B1, EP 494,135 B1, U.S. Pat. No. 5,824,311, EP 444,181 B1, EP 1,006,194 A2, US 2002 / 0155527A1, WO 91 / 02062, U.S. Pat. Nos. 5,571,894, 5,939,531, EP 502,812 B1, WO 93 / 03741, EP 554,441 B1, EP 656,367 A1, U.S. Pat. Nos. 5,288,477, 5,514,554, 5,587,458, WO 93 / 12220, WO 93 / 16185, U.S. Pat. No. 5,877,305, WO 93 / 21319, WO 93 / 21232, U.S. Pat. No. 5,856,089, WO 94 / 22478, U.S. Pat. Nos. 5,910,486, 6,028,059, WO 96 / 07321, U.S. Pat. Nos. 5,804,396, 5,846,749, EP 711,565, WO 96 / 16673, U.S. Pat. Nos. 5,783,404, 5,977,322, 6,512,097, WO 97 / 00271, U.S. Pat. Nos. 6,270,765, 6,395,272, 5,837,243, WO 96 / 40789, U.S. Pat. Nos. 5,783,186, 6,458,356, WO 97 / 20858, WO 97 / 38731, U.S. Pat. Nos. 6,214,388, 5,925,519, WO 98 / 02463, U.S. Pat. No. 5,922,845, WO 98 / 18489, WO 98 / 33914, U.S. Pat. No. 5,994,071, WO 98 / 45479, U.S. Pat. No. 6,358,682 B1, US 2003 / 0059790, WO 99 / 55367, WO 01 / 20033, US 2002 / 0076695 A1, WO 00 / 78347, WO 01 / 09187, WO 01 / 21192, WO 01 / 32155, WO 01 / 53354, WO 01 / 56604, WO 01 / 76630, WO02 / 05791, WO 02 / 11677, U.S. Pat. No. 6,582,919, US2002 / 0192652A1, US 2003 / 0211530A1, WO 02 / 44413, US 2002 / 0142328, U.S. Pat. No. 6,602,670 B2, WO 02 / 45653, WO 02 / 055106, US 2003 / 0152572, US 2003 / 0165840, WO 02 / 087619, WO 03 / 006509, WO03 / 012072, WO 03 / 028638, US 2003 / 0068318, WO 03 / 041736, EP 1,357,132, US 2003 / 0202973, US 2004 / 0138160, U.S. Pat. Nos. 5,705,157, 6,123,939, EP 616,812 B1, US 2003 / 0103973, US 2003 / 0108545, U.S. Pat. No. 6,403,630 B1, WO 00 / 61145, WO 00 / 61185, U.S. Pat. No. 6,333,348 B1, WO 01 / 05425, WO 01 / 64246, US 2003 / 0022918, US 2002 / 0051785 A1, U.S. Pat. No. 6,767,541, WO 01 / 76586, US 2003 / 0144252, WO 01 / 87336, US 2002 / 0031515 A1, WO 01 / 87334, WO 02 / 05791, WO 02 / 09754, US 2003 / 0157097, US 2002 / 0076408, WO 02 / 055106, WO 02 / 070008, WO 02 / 089842 and WO 03 / 86467.

[0018] Patients treated with the HER2 antibody Trastuzumab / Herceptin™ are selected for therapy based on HER2 protein overexpression / gene amplification; see, for example, WO99 / 31140 (Paton et al.), US2003 / 0170234A1 (Hellmann, S.), and US2003 / 0147884 (Paton et al.); as well as WO01 / 89566, US2002 / 0064785, and US2003 / 0134344 (Mass et al.). See, also, US2003 / 0152987, Cohen et al., concerning immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) for detecting HER2 overexpression and amplification. WO2004 / 053497 and US2004 / 024815A1 (Bacus et al.), as well as US 2003 / 0190689 (Crosby and Smith), refer to determining or predicting response to Trastuzumab therapy. US2004 / 013297A1 (Bacus et al.) concerns determining or predicting response to ABX0303 EGFR antibody therapy. WO2004 / 000094 (Bacus et al.) is directed to determining response to GW572016, a small molecule, EGFR-HER2 tyrosine kinase inhibitor. WO2004 / 063709, Amler et al., refers to biomarkers and methods for determining sensitivity to EGFR inhibitor, erlotinib HCl. US2004 / 0209290, Cobleigh et al., concerns gene expression markers for breast cancer prognosis.DETAILED DESCRIPTION

[0019] Patients to be treated with a HER2 dimerization inhibitor (like pertuzumab as described herein above in more detail) can be selected for therapy based on HER activation or dimerization. Patent publications concerning pertuzumab and selection of patients for therapy therewith include: WO01 / 00245 (Adams et al.); US2003 / 0086924 (Sliwkowski, M.); US2004 / 0013667A1 (Sliwkowski, M.); as well as WO2004 / 008099A2, and US2004 / 0106161 (Bossenmaier et al.).

[0020] Herceptin™ / Trastuzumab is indicated in the art for the treatment of patients with metastatic breast cancer whose tumors overexpress HER2 protein or have HER 2 gene amplification:

[0021] a) As monotherapy for the treatment of those patients who have received at least two chemotherapy regimens for their metastatic disease. Prior chemotherapy must have included at least an anthracycline and a taxane unless patients are unsuitable for these treatments. Hormone receptor positive patients must also have received hormonal therapy, unless patients are unsuitable for these treatments,

[0022] b) In combination with paclitaxel for the treatment of those patients who have not received chemotherapy for their metastatic disease and for whom an anthracycline is not suitable and

[0023] c) In combination with docetaxel for the treatment of those patients who have not received chemotherapy for their metastatic disease.

[0024] Herceptin™ / Trastuzumab can also be used as adjuvant treatment in early breast cancer. Herceptin™ / Trastuzumab is also approved for the treatment of patients with HER2-positive early breast cancer following surgery, chemotherapy (neoadjuvant (i.e. before surgery) or adjuvant), and radiotherapy (if applicable). In addition, Herceptin in combination with capecitabine or 5-fluorouracil and cisplatin is indicated for the treatment of patients with HER2 positive locally advance or metastatic adenocarcinoma of the stomach or gastroesophageal junction who have not received prior anti-cancer treatment for their metastatic disease. The efficacy and safety of neoadjuvant pertuzumab and trastuzumab therapy has been assessed in a phase 2 trial (NEOSPHERE); Gianni (2012) Lancet Oncol 13, 25-32.

[0025] In the art, the treatment of breast cancer patients with Herceptin™ / Trastuzumab is, for example, recommended and routine for patients having HER2-positive cancer. HER2-positive cancer is present if a high HER2 (protein) expression level detected by immunohistochemical methods (e.g. HER2 (+++)) or HER2 gene amplification detected by in-situ-hybridization (e.g. ISH positive, like a HER2 gene copy number higher than 4 copies of the HER2 gene per tumor cell or ratio of ≥2.0 for the number of HER2 gene copies to the number of signals for CEP17.) or both is found in samples obtained from the patients such as breast tissue biopsies or breast tissue resections or in tissue derived from metastatic sites.

[0026] WO 2011 / 109789, WO 2011 / 066342, WO 2009 / 089149 and WO2006 / 133396 disclose the therapeutic use of PD-L1 inhibitors. Moreover, WO 2010 / 077634 discloses anti-PD-L1 antibodies and their therapeutic use.

[0027] The present invention relates to a method of determining the need of a cancer patient for a PD-L1 inhibitor cotherapy, (i) wherein therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated for the patient or (ii) wherein the patient is undergoing therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent, said method comprising the steps of

[0028] a) measuring in vitro in a sample from said patient the expression level of Estrogen receptor (ER) and of programmed death ligand 1 (PD-L1),

[0029] b) determining a patient as being in need of a PD-L1 inhibitor cotherapy if a low or absent ER expression level and an expression level of programmed death ligand 1 (PD-L1) that is increased in comparison to a control is measured in step (a).

[0030] Accordingly, the present invention provides a method for determining a cancer patient's need for PD-L1 modulator cotherapy in combination with a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent, the method comprising the steps of

[0031] testing a tumor sample of a patient for whom therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated or who is undergoing said therapy;

[0032] determining the expression level of Estrogen receptor (ER) and of programmed death ligand 1 (PD-L1) in said tumor sample,

[0033] whereby a low or absent ER expression level and an expression level of programmed death ligand 1 (PD-L1) that is increased in comparison to the control is indicative of a successful use of PD-L1 modulator cotherapy in said patient.

[0034] As demonstrated in the appended example, it has been surprisingly found in this invention that Estrogen receptor (ER) negative (ER(−)) cancer patients (cancer patients with a low or even absent ER expression level) undergoing therapy with a modulator of the HER2 / neu (ErbB2) signaling pathway (like Herceptin™ / Trastuzumab) and a chemotherapeutic agent (like dodetaxel / Taxotere®) show a significantly worse pathological complete response (pCR) to the therapy compared to Estrogen receptor (ER) positive (ER(+)) cancer patients, if the expression level of programmed death ligand 1 (PD-L1) is increased in a sample of the ER negative (ER(−)) cancer patients as compared to a control. The terms “programmed death ligand 1”, “CD274” and “PD-L1” are used interchangeably herein. The ER negative (ER(−)) cancer patients with increased expression level of programmed death ligand 1 (PD-L1) as compared to a control will therefore benefit from additional cotherapy with a PD-L1 inhibitor. It is expected that the pathological complete response rate (pCR) in this patient group will increase, if these patients receive cotherapy with a PD-L1 inhibitor in addition to therapy with a modulator of the HER2 / neu (ErbB2) signaling pathway (like Herceptin™ / Trastuzumab) and a chemotherapeutic agent (like dodetaxel / Taxotere®). In other words, the ER negative (ER(−)) cancer patients are to receive a programmed death ligand 1 (PD-L1) inhibitor in addition to a modulator of the HER2 / neu (ErbB2) signaling pathway (like Trastuzumab) and a chemotherapeutic agent (like dodetaxel / Taxotere®), if the expression level of programmed death ligand 1 (PD-L1) is increased in a sample from the patient in comparison to a control. In the following, ER negative cancer patients or (biological / tumor) samples derived from ER negative cancer patients are denoted herein as “ER(−)”. Likewise ER positive cancer patients or (biological / tumor) samples derived from ER positive cancer patients are denoted herein as “ER(+)”.

[0035] In accordance with the above, the present invention relates to a method of treating a cancer in a cancer patient for whom therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated, the method comprising selecting a cancer patient whose cancer is determined to have a low or absent ER expression level and to have an increased expression level of programmed death ligand 1 (PD-L1) in comparison to a control, and administering to the patient an effective amount of a modulator of the HER2 / neu (ErbB2) signaling pathway, of a chemotherapeutic agent and of a programmed death ligand 1 (PD-L1) inhibitor. Likewise, the present invention relates to a method of treating a cancer in a cancer patient who is undergoing therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent, the method comprising selecting a cancer patient whose cancer is determined to have a low or absent ER expression level and to have an increased expression level of programmed death ligand 1 (PD-L1) in comparison to a control, and administering to the patient an effective amount of a programmed death ligand 1 (PD-L1) inhibitor. Herein contemplated is, accordingly, a pharmaceutical composition comprising a modulator of the HER2 / neu (ErbB2) signaling pathway, and an inhibitor of programmed death ligand 1 (PD-L1) for use in the treatment of cancer, whereby said cancer is determined to have a low or absent ER expression level and to have an increased expression level of programmed death ligand 1 (PD-L1) in comparison to a control.

[0036] In accordance with the above, the herein provided method for determining the need of a cancer patient for a PD-L1 inhibitor cotherapy, may comprise an additional step prior to step a), wherein said step is or comprises obtaining a sample from said cancer patient. Accordingly, the present invention provides a method of determining the need of a cancer patient for a PD-L1 inhibitor cotherapy, (i) wherein therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated for the patient or (ii) wherein the patient is undergoing therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent, said method comprising a step of obtaining a sample from said cancer patient, the method further comprising the steps

[0037] a) measuring in vitro in a sample from said patient the expression level of Estrogen receptor (ER) and of programmed death ligand 1 (PD-L1),

[0038] b) determining a patient as being in need of a PD-L1 inhibitor cotherapy if a low or absent ER expression level and an expression level of programmed death ligand 1 (PD-L1) that is increased in comparison to a control is measured in step (a).

[0039] Furthermore, it has been found herein and is demonstrated in the appended example, that a patient's need of PD-L1 inhibitor cotherapy can be determined even more reliably, if the expression level of interferon-gamma (IFNγ) is measured in the sample of the patient in addition to the expression level of programmed death ligand 1 (PD-L1). It is shown herein that patients with low or absent ER expression have a significantly worse pathologic complete response to therapy with a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent, if the expression level of programmed death ligand 1 (PD-L1) is increased and if the expression level of interferon-gamma (IFNγ) is decreased.

[0040] Accordingly, the methods provided herein preferably further comprise measuring the expression level of interferon-gamma (IFNγ) in the sample from the patient, whereby a patient is determined to be in need of a PD-L1 inhibitor cotherapy, if the expression level of interferon-gamma (IFNγ) is decreased in comparison to a control. In accordance with the above, the present invention relates in a preferred aspect to a method of determining the need of a cancer patient for a PD-L1 inhibitor cotherapy, (i) wherein therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated for the patient or (ii) wherein the patient is undergoing therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent, said method comprising the steps of

[0041] a) measuring in vitro in a sample from said patient the expression level of Estrogen receptor (ER), the expression level of programmed death ligand 1 (PD-L1), and the expression level of interferon-gamma (IFNγ)

[0042] b) determining a patient as being in need of a PD-L1 inhibitor cotherapy if a low or absent ER expression level, an expression level of programmed death ligand 1 (PD-L1) that is increased in comparison to a control, and an expression level of interferon-gamma (IFNγ) that is decreased in comparison to a control is measured in step (a).

[0043] Accordingly, an expression level of interferon-gamma (IFNγ) that is decreased in comparison to a control is indicative of a successful use of PD-L1 inhibitor cotherapy in said patient. The herein provided pharmaceutical composition is, in accordance with the above, for use in the treatment of cancer, whereby said cancer is determined to have a low or absent ER expression level, the cancer is determined to have an increased expression level of programmed death ligand 1 (PD-L1) in comparison to a control and the cancer is determined to have a decreased expression level of interferon-gamma (IFNγ) in comparison to the control. Accordingly, a pharmaceutical composition is provided herein comprising a modulator of the HER2 / neu (ErbB2) signaling pathway, and an inhibitor of programmed death ligand 1 (PD-L1) for use in the treatment of cancer, whereby said cancer is determined to have a low or absent ER expression level and to have an increased expression level of programmed death ligand 1 (PD-L1) in comparison to a control and to have a decreased expression level of interferon-gamma (IFNγ) in comparison to the control.

[0044] The term “cancer patient” as used herein refers to a patient that is suspected to suffer from cancer, suffering from cancer or being prone to suffer from cancer. The cancer to be treated in accordance with the present invention can be a solid cancer, such as breast cancer or gastric cancer. Further, the cancer may be ovarian cancer or colorectal cancer. The cancer is preferably a “HER2-positive” cancer.

[0045] Preferably, the cancer is breast cancer, like early breast cancer. The breast cancer may be early stage breast cancer or metastatic breast cancer. Accordingly, the cancer patient (to be treated) is suspected to suffer from solid cancer, is suffering from solid cancer or is being prone to suffer from solid cancer, whereby the solid cancer can be breast cancer or gastric cancer. Preferably, the cancer is breast cancer, like early stage breast cancer. The patient is preferably a human.

[0046] As mentioned above, the expression level of Estrogen receptor (ER) and of programmed death ligand (PD-L1), and optionally of interferon-gamma (IFN-γ) can be measured in vitro in a sample from the patient. Preferably, the herein provided methods comprise measuring of interferon-gamma (IFN-γ) in vitro in a sample from the patient. Preferably, the sample to be assessed / analyzed herein is a tumor tissue sample. A patient (or a patient group) is determined as being in need of a PD-L1 inhibitor cotherapy if a low or absent ER expression level and an expression level of programmed death ligand 1 (PD-L1) that is increased in comparison to a control and, optionally, an expression level of interferon-gamma (IFNγ) that is decreased in comparison to the control, is measured in vitro in said sample.

[0047] The term “ER” is an abbreviation of “Estrogen receptor”. Likewise, the terms “PD-L1” and “IFN-γ” are abbreviations of the terms “programmed death ligand” and “interferon-gamma”, respectively. Accordingly, the term “ER” can be used interchangeably herein with “Estrogen receptor”. Likewise, the terms “PD-L1” and “IFN-γ” can be used interchangeably herein with the terms “programmed death ligand” and “interferon-gamma”, respectively.

[0048] Preferably, the (tumor / biological) sample of the patient and / or the cancer to be treated is characterized by or associated with a low or absent estrogen receptor (ER) expression level. Preferably, the sample of the patient is a tumor sample. The ER expression level can be ER negative (ER(−)). The term “ER(−)” can be used herein interchangeably with the term “ER negative”.

[0049] “ER negative” expression level can be determined by routine and standard procedures as described, for example, in the Guideline on Hormone Receptor Testing in Breast Cancer S. Nofech-Mozes, E. Vella, S. Dhesy-Thind, and W. Hanna (A Quality Initiative of the Program in Evidence-Based Care (PEBC), Cancer Care Ontario (CCO); Report Date: Apr. 8, 2011). The Guidelines (and references cited therein) are incorporated by reference in its entirety herein. These Guidelines are available at world wide web atcancercare.on.ca) andPEBC Pathology & Laboratory Medicine page at:cancercare.on.ca / toolbox / qualityguidelines / clin-program / pathlabebs /

[0050] Routine and standard procedures for determining the “ER negative” expression level are described in these Guideline and also in the following references:

[0051] Nofech-Mozes S, Vella E T, Dhesy-Thind S, Hagerty K L, Mangu P B, Temin S, et al. Systematic review on hormone receptor testing in breast cancer. Applied Immunohistochem Mol Morphol. 2012 May; 20(3):214-63. doi: 10.1097 / PAI.0b013e318234aa12. Epub 2011 Nov. 11.

[0052] Nofech-Mozes S, Vella E T, Dhesy-Thind S, Hanna W M. Cancer Care Ontario guideline recommendations for hormone receptor testing in breast cancer. Clin Oncol (R Coll Radiol). Epub 2012 May 17.

[0053] “ER negative” expression may be determined by IHC (immunohistochemistry), if, for example the expression level of ER is low or absent and / or if the progesterone receptor (PR) expression level is low or absent. The abbreviation “PR” is used herein interchangeably with the term “progesterone receptor”. A sample or patients may be assessed as “ER negative” herein according to the following staining pattern (by IHC):

[0054] Only nuclear (not cytoplasmic) staining should be scored.

[0055] There are three categories for staining:

[0056] Positive: >10% staining for ER or PR

[0057] Low positive: 1% to 9% staining for ER or PR

[0058] Negative: <1% staining for ER and PR

[0059] Accordingly, a sample or patients may particularly be assessed as “ER negative” herein if the sample shows the following staining pattern by IHC: <1% staining for ER and PR.

[0060] Samples or patients may be assessed as “ER positive” herein if the sample shows a “positive” staining by IHC: ≥1% staining for ER or PR (i.e. more than 1% of the cells examined / assessed have estrogen receptors or progesterone receptors / show staining for estrogen receptors by IHC (immunohistochemistry).

[0061] Preferably, a sample or patient is assessed as “ER negative” herein if the sample shows the following staining pattern by IHC::<1% staining for ER (i.e. less than 1% of the cells examined / assessed have estrogen receptors / show staining for estrogen receptor(s) by IHC (immunohistochemistry). Most preferably, a sample or patients is / are assessed as “ER negative” if the nuclei in a tumor tissue sample show <1% staining for ER staining by IHC. Accordingly, from the three categories provided herein above, the assessment of “ER negative” is based on <1% staining for ER by IHC.

[0062] Likewise, “ER negative” expression can be determined by further methods routinely employed in the art. For example, “ER negative” may be determined if the mRNA / RNA expression level is low or absent. Routine methods to be used comprise, but are not limited to: Allred score, IRS, Remmele score or any other suitable biochemical detection method. A person skilled in the art is aware that the cut-off for such methods has to match the cut-off as defined above via IHC.

[0063] Nucleic acid sequences and amino acid sequences of Progesterone receptor (PR), Estrogen receptor (ER), of programmed death ligand 1 (PD-L1), and / or of interferon-gamma (IFNγ) to be used herein are well known and can be retrieved from databases like NCBI. Examplary sequences are provided herein (see for example SEQ ID NO: 38-51).

[0064] The methods and sample types used for establishing a cut-off value of a marker (like programmed death ligand 1 (PD-L1) and / or interferon-gamma (IFN-γ)) and for measuring the sample obtained from an individual or patient to be analyzed match each other or are the same. Cut-off values, i.e. values above which overexpression (e.g. increased expression of programmed death ligand 1 (PD-L1) in comparison to a control) is acknowledged can be obtained in a control group. Cut-off values, i.e. values below which decreased expression (e.g. decreased expression of interferon-gamma (IFN-7) in comparison to a control) is acknowledged can be obtained in a control group.

[0065] The control group on which the cut-off value is based is chosen to match the group of individuals / patients under investigation. In other words, if the method of the present invention is used to determine the need for PD-L1 cotherapy in patients with breast cancer or gastric cancer, respectively, the control group is also patients with breast cancer or gastric cancer, respectively. The control group used to establish the cut-off values for both, PDL-1 and IFN-γ, respectively), comprises at least 40, or at least 50, or at least 100 individuals / patients. An expression level or corresponding value above the cut-off is considered to represent overexpression and a value at or below the cut-off is considered as decreased expression.

[0066] In one embodiment, the “IFN-γ” expression level in a tumor tissue sample from an individual / patient is compared to a cut-off value. A value above the cut-off is considered to represent overexpression of IFN-γ and a value at or below the cut-off is considered as decreased expression of IFN-γ. In one embodiment the decreased expression is acknowledged if the expression level for IFN-γ is at or below the value of the highest quintile, quartile or tertile, respectively, as established in the control group. In one embodiment the cut-off for IFN-γ is the highest tertile. In one embodiment the cut-off value is a value between the 70th and the 80th percentile. In one embodiment the cut-off value for IFN-γ is the 73rd percentile, i.e a value above this cut-off is considered to represent overexpression of IFN-γ and a value at or below the 73rd percentile is considered as decreased expression of IFN-γ. In one embodiment, individuals / patients are determined as being in need of a PD-L1 cotherapy, if IFN-γ expression in a sample (like a tumor tissue sample) is decreased (i.e. below or at the IFN-γ cut-off value) In one embodiment individuals / patients are determined as not being in need of a PDL-1 cotherapy, if IFN-γ is overexpressed (i.e. above the IFN-γ cut-off value as described above).

[0067] In one embodiment the PD-L1 expression level, in a tumor tissue sample from an individual / patient is compared to a cut-off value. A value above the cut-off is considered to represent overexpression of PD-L1 and a value at or below the cut-off is considered as decreased expression of PD-L1. In one embodiment overexpression for PDL-1 is acknowledged if the expression level for PDL-1 is above a cut-off value between the 50th percentile and the 75th percentile, as established in a control group. In one embodiment overexpression for PDL-1 is acknowledged if the expression level for PDL-1 is above a cut-off value between the 50th percentile and the 70th percentile, of the control group. In one embodiment individuals / patients are determined as being in need of a PDL-1 cotherapy, if PDL-1 is overexpressed (i.e. the PDL-1 expression level determined is above the PDL-1 cut-off value).

[0068] In one further embodiment overexpression for PDL-1 is established in the sub-group of individuals / patients having a decreased expression level of IFN-γ in a tumor tissue sample. In one embodiment overexpression for PDL-1 is acknowledged if the expression level for PDL-1 is above a cut-off value between the 40th percentile and the 65th percentile, as established in this sub-group. In one embodiment overexpression for PDL-1 is acknowledged if the expression level for PDL-1 is above a cut-off value between the 50th percentile and the 60th percentile, as established in this sub-group. In one embodiment individuals / patients are determined as being in need of a PDL-1 cotherapy, if the PDL-1 expression level in the sub-group with decreased expression of IFN-γ is above the 54th percentile.

[0069] In one embodiment, individuals / patients are determined as being in need of a PDL-1 cotherapy, if IFN-γ expression in a tumor tissue sample is decreased (i.e. below or at the IFN-γ cut-off value) and PDL-1 is overexpressed (i.e. above the PDL-1 cut-off value).

[0070] The term “expression level of programmed death ligand 1 (PD-L1) that is increased in comparison to a control” can be used interchangeably herein with “expression level of programmed death ligand 1 (PD-L1) above the PDL-1 cut-off value” as defined and explained herein above.

[0071] The term “expression level of interferon-gamma (IFNγ) that is decreased in comparison to a control” can be used interchangeably herein with “expression level of interferon-gamma (IFNγ) below or at the IFNγ cut-off value”.

[0072] The present invention relates to the following aspects.

[0073] The present invention relates to a method of determining the need of a cancer patient for a PD-L1 inhibitor cotherapy, (i) wherein therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated for the patient or (ii) wherein the patient is undergoing therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent, the method comprising the steps of

[0074] a) measuring in vitro in a sample from said patient the expression level of Estrogen receptor (ER), of programmed death ligand 1 (PD-L1), and of interferon-gamma (IFNγ);

[0075] b) determining a patient as being in need of a PD-L1 inhibitor cotherapy if a low or absent ER expression level (like ER(−) / ER-negative), an expression level of programmed death ligand 1 (PD-L1) above the PDL-1 cut-off value and an expression level of interferon-gamma (IFNγ) below or at the IFNγ cut-off value is measured in step (a).

[0076] The present invention relates to a method of treating a cancer in a cancer patient for whom therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated, the method comprising selecting a cancer patient whose cancer is determined to have a low or absent ER expression level (like ER(−) / ER-negative) and to have an expression level of programmed death ligand 1 (PD-L1) above the PDL-1 cut-off value and to have an expression level of interferon-gamma (IFNγ) below or at the IFNγ cut-off value, and administering to the patient an effective amount of a modulator of the HER2 / neu (ErbB2) signaling pathway, of a chemotherapeutic agent and of a programmed death ligand 1 (PD-L1) inhibitor.

[0077] The present invention relates to a method of treating a cancer in a cancer patient who is undergoing therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent, the method comprising selecting a cancer patient whose cancer is determined to have a low or absent ER expression level (like ER(−) / ER-negative) and to have an expression level of programmed death ligand 1 (PD-L1) above the PDL-1 cut-off value and to have an expression level of interferon-gamma (IFNγ) below or at the IFNγ cut-off value, and administering to the patient an effective amount of a programmed death ligand 1 (PD-L1) inhibitor.

[0078] The present invention relates to a pharmaceutical composition comprising a modulator of the HER2 / neu (ErbB2) signaling pathway, and an inhibitor of programmed death ligand 1 (PD-L1) for use in the treatment of cancer, whereby said cancer is determined to have a low or absent ER expression level (like ER(−) / ER-negative) and to have an expression level of programmed death ligand 1 (PD-L1) above the PDL-1 cut-off value and to have an expression level of interferon-gamma (IFNγ) below or at the IFNγ cut-off value.

[0079] All explanations and definitions given herein for “PD-L1 inhibitor”, “PD-L1 inhibitor cotherapy”, “cancer”, “cancer patient”, “modulator of the HER2 / neu (ErbB2) signaling pathway”, “chemotherapeutic agent”, “sample”, “expression level” and the like apply, mutatis mutandis, to the above aspects of the present invention.

[0080] The expression level of Estrogen receptor (ER), of programmed death ligand 1 (PD-L1), and of interferon-gamma (IFNγ) in a sample from the patient may be measured in vitro simultaneously or subsequently in any combination. For example, the expression level of Estrogen receptor (ER), of programmed death ligand 1 (PD-L1), and of interferon-gamma (IFNγ) may be measured simultaneously. The expression level of Estrogen receptor (ER) may be measured first, followed by the measurement of programmed death ligand 1 (PD-L1) and of interferon-gamma (IFNγ). The expression level of programmed death ligand 1 (PD-L1) may be measured first, followed by the (simultaneous or subsequent) measurement of Estrogen receptor (ER) and of interferon-gamma (IFNγ). The expression level of interferon-gamma (IFNγ) may be measured first, followed by the (simultaneous or subsequent) measurement of Estrogen receptor (ER) and of programmed death ligand 1 (PD-L1). Any order / combination of the measurement of the expression level of Estrogen receptor (ER), of programmed death ligand 1 (PD-L1), and of interferon-gamma (IFNγ) in a sample from the patient is envisaged and comprised herein.

[0081] Herein contemplated is a determination of a patient as being in need of a PD-L1 inhibitor cotherapy if, in a first step (1) a low or absent ER expression level (like ER(−) / ER-negative) is measured, and if, in a second step (2) an expression level of interferon-gamma (IFNγ) below or at the IFNγ cut-off value is measured and if, in a third step (3) an expression level of programmed death ligand 1 (PD-L1) above the PDL-1 cut-off value is measured.

[0082] The present invention relates to the following aspects:

[0083] The present invention relates to a method of determining the need of a cancer patient for a PD-L1 inhibitor cotherapy, (i) wherein therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated for the patient or (ii) wherein the patient is undergoing therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent, the method comprising the steps of

[0084] a) measuring in vitro in a sample from said patient the expression level of Estrogen receptor (ER), of programmed death ligand 1 (PD-L1), and of interferon-gamma (IFNγ);

[0085] b) determining a patient as being in need of a PD-L1 inhibitor cotherapy if, in a first step (1) a low or absent ER expression level (like ER(−) / ER-negative) is measured, and if in a second step (2) an expression level of interferon-gamma (IFNγ) below or at the IFNγ cut-off value is measured and if in a third step (3) an expression level of programmed death ligand 1 (PD-L1) above the PDL-1 cut-off value is measured.

[0086] The present invention relates to a method of treating a cancer in a cancer patient for whom therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated, the method comprising selecting a cancer patient whose cancer is determined to have in a first step (1) a low or absent ER expression level (like ER(−) / ER-negative) and in a second step (2) to have an expression level of interferon-gamma (IFNγ) below or at the IFNγ cut-off value, and in a third step (3) to have an expression level of programmed death ligand 1 (PD-L1) above the PDL-1 cut-off value, and administering to the patient an effective amount of a modulator of the HER2 / neu (ErbB2) signaling pathway, of a chemotherapeutic agent and of a programmed death ligand 1 (PD-L1) inhibitor.

[0087] The present invention relates to a method of treating a cancer in a cancer patient who is undergoing therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent, the method comprising selecting a cancer patient whose cancer is determined to have in a first step (1) a low or absent ER expression level (like ER(−) / ER-negative) and in a second step (2) to have an expression level of interferon-gamma (IFNγ) below or at the IFNγ cut-off value, and in a third step (3) to have an expression level of programmed death ligand 1 (PD-L1) above the PDL-1 cut-off value, and administering to the patient an effective amount of a programmed death ligand 1 (PD-L1) inhibitor.

[0088] The present invention relates to a pharmaceutical composition comprising a modulator of the HER2 / neu (ErbB2) signaling pathway, and an inhibitor of programmed death ligand 1 (PD-L1) for use in the treatment of cancer, whereby said cancer is determined to have a low or absent ER expression level (like ER(−) / ER-negative), to have an expression level of interferon-gamma (IFNγ) below or at the IFNγ cut-off value, to have an expression level of programmed death ligand 1 (PD-L1) above the PDL-1 cut-off value.

[0089] All explanations and definitions given herein for “PD-L1 inhibitor”, “PD-L1 inhibitor cotherapy”, “cancer”, “cancer patient”, “modulator of the HER2 / neu (ErbB2) signaling pathway”, “chemotherapeutic agent”, “sample”, “expression level” and the like apply, mutatis mutandis, to the above aspects of the present invention.

[0090] The following relates to an exemplary cut-off value allowing determining a patient as being in need of a PD-L1 inhibitor cotherapy in accordance with the present invention. It can be easily determined by routine techniques (such as Affymetrix) whether the expression level of PD-L1 and / or IFN-gamma in a sample from a patient is below or above such cut-off values.

[0091] If a gene expression analysis gives a result for IFN-gamma expression higher or equal to 4.8 no combination treatment (HER2-targeted and PDL1-targeted) is recommended and no further PDL1 assessment is necessary. If a gene expression analysis gives a result for IFN-gamma lower than 4.8 a parallel assessment of PDL-1 is necessary. If PDL-1 gene expression analysis then gives a result of higher or equal to 5.3 a combination treatment (HER2-targeted and PDL1-targeted) is recommended. This exemplary protocol is illustrated in FIG. 19.

[0092] In this context Affymetrix can be performed as follows: Total RNA from tumor cells was extracted FFPE tumor sections using Light Cycler Pertuzumab FFPET RNA Kit (Roche Diagnostics). RNA was processed for hybridization using the WT-Ovation FFPE System V2 (Nugen) and hybridized to Affymetrix GeneChip® Human Genome U133 Plus 2.0 Arrays. Hybridized arrays were washed and stained on Affymetrix Fluidics Station 450 and scanned with an Affymetrix GeneChip® Scanner 3000 7G.

[0093] As mentioned the expression level of PD-L1 and / or IFN-gamma in a sample from a patient can be determined by routine techniques, such as Affymetrix. The following relates an exemplary protocol for such a determination (also termed herein Gene Expression Profiling):

[0094] The tumor biopsy samples can be profiled for gene expression on AFFYMETRIX HG-U133Plus 2 whole Human Genome microarray platform. Roche HighPure RNA extraction, NuGen amplification and standard AFFYMETRIX hybridization and scanning protocols can be used. These protocols etc. are incorporated herein by reference. All array scans usually pass standard AFFYMETRIX QC.

[0095] Robust Multiarray algorithm (RMA) can be used for preprocessing of raw signals (Irizarry et al, 2003. World wide web at ncbi.nlm.nih.gov / pubmed / 12925520; incorporated herein by reference). All probe sets available for the genes of interest can be retrieved as reported below. For gene CD274, when several probe sets were available to represent this gene, the probe set with the highest average expression value (defined as an arithmetical average of expression of a given probe set) was selected to represent the gene:

[0096] CD274 (PDL1)

[0097] 223834_at selected for PDL1

[0098] 227458_at

[0099] The selected probe set corresponds to the last exon / 3′UTR of the gene and captures all known RefSEq mRNAs (see FIG. 6)

[0100] IFNG

[0101] 210354_at

[0102] This probe set also represents the last exon / 3′UTR of the gene and captures all known RefSEq mRNAs (see FIG. 7)

[0103] In accordance with the above, the expression level of Interferon-gamma may be measured prior to the expression level of Estrogen receptor (ER) and / or prior to the expression level of programmed death ligand 1 (PD-L1). The step of measuring the expression level of Estrogen receptor (ER) and of programmed death ligand 1 (PD-L1) may even be absent.

[0104] As shown in the appended Example, PD-L1 cotherapy can, for example, not be recommended if the expression level of interferon-gamma (IFNγ) is higher or equal to (about) 4.8 as determined by routine methods like Affymetrix.

[0105] Accordingly, the present invention provides a method of determining the need of a cancer patient for a PD-L1 inhibitor cotherapy, wherein therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated for the patient or wherein the patient is undergoing therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent, the method comprising the steps

[0106] (a) measuring in vitro in a sample from said patient the expression level of interferon-gamma (IFNγ)

[0107] (b) determining a patient as being not in need of a PD-L1 inhibitor cotherapy if the expression level of interferon-gamma (IFNγ) is higher or equal to (about) 4.8 as determined by routine methods like Affymetrix in step (a).

[0108] If the expression level of interferon-gamma (IFNγ) is lower than (about) 4.8 as determined by routine methods like Affymetrix, the expression level of programmed death ligand 1 (PD-L1) and, optionally, Estrogen receptor (ER) can be measured in vitro in a sample from said patient.

[0109] Accordingly, the present invention provides a method of determining the need of a cancer patient for a PD-L1 inhibitor cotherapy, wherein therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated for the patient or wherein the patient is undergoing therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent, the method comprising the steps

[0110] (a) measuring in vitro in a sample from said patient the expression level of interferon-gamma (IFNγ), Estrogen receptor (ER) and of programmed death ligand 1 (PD-L1),

[0111] (b) determining a patient as being in need of a PD-L1 inhibitor cotherapy if the expression level of interferon-gamma (IFNγ) is lower than (about) 4.8 as determined by routine methods like Affymetrix, and if a low or absent ER expression level and, optionally, an expression level of programmed death ligand 1 (PD-L1) that is increased in comparison to a control is measured in step (a).

[0112] A patient can be determined in accordance with the present invention to be in need of PD-L1 inhibitor cotherapy if the expression level of programmed death ligand 1 (PD-L1) measured in the sample from the patient is increased in comparison to a control. For example, the expression level of programmed death ligand 1 (PD-L1) can be higher or equal to (about) 5.3 determined by routine methods like Affymetrix.

[0113] All explanations and definitions given herein for “PD-L1 inhibitor”, “PD-L1 inhibitor cotherapy”, “cancer”, “cancer patient”, “modulator of the HER2 / neu (ErbB2) signaling pathway”, “chemotherapeutic agent”, “sample”, “expression level” and the like as given herein apply, mutatis mutandis, in this context.

[0114] Accordingly, the present invention relates to a method of treating a cancer in a cancer patient for whom therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated, the method comprising selecting a cancer patient whose cancer is determined to have a low or absent ER expression level and to have an increased expression level of programmed death ligand 1 (PD-L1) in comparison to a control, and an expression level of interferon-gamma (IFNγ) that is lower than (about) 4.8 as determined by routine methods like Affymetrix, and administering to the patient an effective amount of a modulator of the HER2 / neu (ErbB2) signaling pathway, of a chemotherapeutic agent and of a programmed death ligand 1 (PD-L1) inhibitor.

[0115] Furthermore, the present invention relates to a method of treating a cancer in a cancer patient who is undergoing therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent, the method comprising selecting a cancer patient whose cancer is determined to have a low or absent ER expression level and to have an increased expression level of programmed death ligand 1 (PD-L1) in comparison to a control, and to have an expression level of interferon-gamma (IFNγ) that is lower than (about) 4.8 as determined by routine methods like Affymetrix, and administering to the patient an effective amount of a programmed death ligand 1 (PD-L1) inhibitor.

[0116] A pharmaceutical composition is provided comprising a modulator of the HER2 / neu (ErbB2) signaling pathway, and an inhibitor of programmed death ligand 1 (PD-L1) for use in the treatment of cancer, whereby said cancer is determined to have a low or absent ER expression level and to have an increased expression level of programmed death ligand 1 (PD-L1) in comparison to a control, and an expression level of interferon-gamma (IFNγ) that is lower than (about) 4.8 as determined by routine methods like Affymetrix.

[0117] The pharmaceutical composition for use in the treatment of cancer may further comprise a chemotherapeutic agent.

[0118] In accordance with the above, the herein provided methods may comprise a step of measuring the expression level of Interferon-gamma (IFNγ) in said sample and determining a patient as being in need of a PD-L1 inhibitor cotherapy if an expression level of interferon-gamma (IFNγ) that is decreased in comparison to the control is measured. For example, a “decreased expression level” of interferon-gamma (IFNγ) may be an expression level lower than (about) 4.8 as determined by routine methods like Affymetrix. Accordingly, the cancer that is determined to have a decreased expression level of interferon-gamma (IFNγ) in comparison to the control may be determined to have an expression level of interferon-gamma (IFNγ) that is lower than (about) 4.8 as determined by routine methods like Affymetrix,

[0119] It is envisaged herein that the expression level may be reflected in the activity of the gene product / protein. Accordingly, also the activity of ER, PD-L1 and / or IFN-γ can be measured and evaluated in addition or in the alternative to the expression level in accordance with the present invention. A person skilled in the art is aware of corresponding means and methods for detecting and evaluating the ER, PD-L1 and IFN-γ expression level and / or activity. Exemplary methods to be used include but are not limited to molecular assessments such as Western Blots, Northern Blots, Real-Time PCR and the like. Such methods are described herein in detail.

[0120] The expression level of ER, PD-L1 and / or IFN-γ may be the mRNA expression level of ER, PD-L1 and / or IFN-γ. If the gene product is an RNA, in particular an mRNA (e.g. unspliced, partially spliced or spliced mRNA), determination can be performed by taking advantage of northern blotting techniques, in situ hybridization, hybridization on microarrays or DNA chips equipped with one or more probes or probe sets specific for mRNA transcripts or PCR techniques, like, quantitative PCR techniques, such as Real time PCR. These and other suitable methods for binding (specific) mRNA are well known in the art and are, for example, described in Sambrook and Russell (2001, loc. cit.). A skilled person is capable of determining the amount of the component, in particular said gene products, by taking advantage of a correlation, preferably a linear correlation, between the intensity of a detection signal and the amount of the gene product to be determined.

[0121] The expression level may be the protein expression level of ER, PD-L1 and / or IFN-γ. Quantification of the protein expression level can be performed by taking advantage of the well known techniques such as western blotting techniques, immunoassays, gel- or blot-based methods, IHC, mass spectrometry, flow cytometry, FACS and the like. Generally, a person skilled in the art is aware of methods for the quantitation of (a) polypeptide(s) / protein(s). Amounts of purified polypeptide in solution can be determined by physical methods, e.g. photometry. Methods of quantifying a particular polypeptide in a mixture may rely on specific binding, e.g of antibodies. Specific detection and quantitation methods exploiting the specificity of antibodies comprise for example immunohistochemistry (in situ). Western blotting combines separation of a mixture of proteins by electrophoresis and specific detection with antibodies. Electrophoresis may be multi-dimensional such as 2D electrophoresis. Usually, polypeptides are separated in 2D electrophoresis by their apparent molecular weight along one dimension and by their isoelectric point along the other direction. Alternatively, protein quantitation methods may involve but are not limited to mass spectrometry or enzyme-linked immunosorbant assay methods.

[0122] Also, the use of high throughput screening (HTS) is envisaged in the context of the present invention. Suitable (HTS) approaches are known in the art. A person skilled in the art is readily in the position to adapt such protocols or known HTS approaches to the performance of the methods of the present invention. Such assays are usually performed in liquid phase, wherein for each cell / tissue / cell culture to be tested at least one reaction batch is made. Typical containers to be used are micro titer plates having for example, 384, 1536, or 3456 wells (i.e. multiples of the “original” 96 reaction vessels). Robotics, data processing and control software, and sensitive detectors, are further commonly used components of a HTS device. Often robot systems are used to transport micro titer plates from station to station for addition and mixing of sample(s) and reagent(s), incubating the reagents and final readout (detection). Usually, HTS can be used in the simultaneous preparation, incubation and analysis of many plates. The assay can be performed in a single reaction (which is usually preferred), may, however, also comprise washing and / or transfer steps. Detection can be performed taking advantage of radioactivity, luminescence or fluorescence, like fluorescence-resonance-energy transfer (FRET) and fluorescence polarisation (FP) and the like. The biological samples described herein can also be used in such a context. In particular, cellular assays and in vivo assays can be employed in HTS. Cellular assays may also comprise cellular extracts, i.e. extracts from cells, tissues and the like. However, preferred herein is the use of cell(s) or tissue(s) as biological sample (in particular a sample obtained from a patient / subject suffering or being prone to suffer from cancer), whereas in vivo assays are particularly useful in the validation of modulators / inhibitors / chemotherapeutic agents to be used herein. Depending on the results of a first assay, follow up assays can be performed by re-running the experiment to collect further data on a narrowed set (e.g. samples found “positive” in the first assay), confirming and refining observations.

[0123] As used in context of the methods of the present invention, a non-limiting example of a “control” is preferably a control from a patient who is not in need of a PD-L1 inhibitor cotherapy, for example a sample / cell / tissue obtained from one or more healthy subjects or one or more patients that suffer from a cancer / tumor and are known to be not in need of a PD-L1 inhibitor cotherapy treatment. For example, such a control (sample) may be from a patient who does not benefit from additional PD-L1 inhibitor cotherapy. Another non-limiting example of a “control” is an “internal standard”, for example a mixture of purified or synthetically produced proteins and / or peptides or RNA, where the amounts of each protein / peptide / RNA is gauged by using the control described above.

[0124] A further non-limiting example of a “control” may be a “healthy” control, for example a sample / cell / tissue obtained from a healthy subject or patient that is not suffering from a cancer / tumor or a cell obtained from such a subject. In accordance with the above, the reference or control expression level of ER, PD-L1 and / or IFN-γ is that determined in (a sample of) the corresponding healthy control subject / patient, i.e. it is the “normal” status of ER, PD-L1 and / or IFN-γ. The control may also be a sample / cell / tissue obtained from the individual or patient suspected of suffering from the cancer provided that the sample / cell / tissue does not contain tumor or cancer cells. In a further alternative, the “control” may be a sample / cell / tissue obtained from an individual or patient suffering from the cancer, that has been obtained prior to the development or diagnosis of said cancer.

[0125] The sample to be assessed in accordance with the herein provided methods may comprise non-diseased cells and / or diseased cells, i.e. non-cancerous cells and / or cancerous cells. However, the content of cancerous cells among non-cancerous cells should be higher than for example 50%. The sample may also (or even solely) comprise cancer / tumor cell(s), such as breast cancer / tumor cell(s). The term “sample” shall generally mean any biological sample obtained from a patient's tumor. The sample may be a tissue resection or a tissue biopsy. The sample may also be a metastatic lesion or a section of a metastatic lesion or a blood sample known or suspected to comprise circulating tumor cells. In accordance with the above, the biological sample may comprise cancer cells and to a certain extent i.e. less than for example 50% non-cancer cells (other cells). The skilled pathologist is able to differentiate cancer cells from normal tissue cells. Methods for obtaining tissue biopsies, tissue resections and body fluids and the like from mammals, such as humans, are well known in the art.

[0126] As explained above, the cancer patient who is determined to be in need of PD-L1 inhibitor cotherapy in accordance with the present invention is undergoing therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent or such a therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated for the patient. Therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent is indicated for patients with “HER2-positive cancer”, like a patient that is suspected to suffer from a HER2-positive cancer, suffering from a HER2-positive cancer or being prone to suffer from a HER2-positive cancer. Preferably, the cancer to be treated is in accordance with the present invention a “HER2-positive cancer”, particularly a “HER2-positive breast cancer”. A “HER2-positive cancer” can be a “HER2-positive breast cancer” or a “HER2-positive gastric cancer”. Further, the HER2-positive cancer may be ovarian cancer, lung cancer, colorectal cancer, kidney cancer, bone cancer, bone marrow cancer, bladder cancer, skin cancer, prostate cancer, esophagus cancer, salivary gland cancer, pancreas cancer, liver cancer, head and neck cancer, CNS (especially brain) cancer, cervix cancer, cartilage cancer, colon cancer, genitourinary cancer, gastrointestinal tract cancer, pancreas cancer, synovium cancer, testis cancer, thymus cancer, thyroid cancer and uterine cancer.

[0127] The term “HER2-positive cancer” as used herein refers to a cancer / tumorous tissue etc. which comprises cancer cells which have higher than normal levels of HER2. For the purpose of the present invention, “HER2-positive cancer” has an immunohistochemistry (IHC) score of at least 2+ and / or an in situ hybridization (ISH) amplification ratio ≥2.0 (i.e. is ISH-positive). Accordingly, HER2-positive cancer is present if a high HER2 (protein) expression level detected e.g. by immunohistochemical methods and / or HER2 gene amplification detected by in-situ-hybridization (ISH positive, like a HER2 gene copy number higher than 4 copies of the HER2 gene per tumor cell or ratio of ≥2.0 for the number of HER2 gene copies to the number of signals for CEP17.) is found in samples obtained from the patients such as breast tissue biopsies or breast tissue resections or in tissue derived from metastatic sites. In one embodiment “HER2-positive cancer” has an immunohistochemistry (IHC) score of HER2(3+) and / or is ISH positive.

[0128] The expression level of HER2 may be detected by an immunohistochemical method, whereas said HER2 gene amplification status can be measured with in situ hybridization methods, like fluorescence in situ hybridization techniques (FISH). Corresponding assays and kits are well known in the art, for protein expression assays as well as for the detection of gene amplifications. Alternatively, other methods like qRT-PCR might be used to detect levels of HER2 gene expression.

[0129] The expression level of HER2 can, inter alia, be detected by an immunohistochemical method. Such methods are well known in the art and corresponding commercial kits are available. Exemplary kits which may be used in accordance with the present invention are, inter alia, HerceptTest™ produced and distributed by the company Dako or the test called Ventana Pathway™. The level of HER2 protein expression may be assessed by using the reagents provided with and following the protocol of the HercepTest™. A skilled person will be aware of further means and methods for determining the expression level of HER2 by immunohistochemical methods; see for example WO 2005 / 117553. Therefore, the expression level of HER2 can be easily and reproducibly determined by a person skilled in the art without undue burden. However, to ensure accurate and reproducible results, the testing must be performed in a specialized laboratory, which can ensure validation of the testing procedures. The expression level of HER2 can be classified in a low expression level, an intermediate expression level and a high expression level. It is preferred in context of this invention that HER2-positive disease is defined by a strong expression level of HER2 (e.g. HER2(3+) by IHC), for example determined in a sample of a cancer patient.

[0130] The recommended scoring system to evaluate the IHC staining patterns which reflects the expression levels of HER2 designated herein HER2(0), HER2(+), HER2(++) and HER2(+++), is as follows:StainingHER2IntensityoverexpressionScoreStaining Patternassessment0 No staining is observed or negativemembrane staining is observedin <10% of the tumor cells1+A faint / barely perceptible negativemembrane staining is detectedin >10% of the tumor cells, the cells are only stained inpart of their membrane.2+A weak to moderate complete weak to staining is detected in >10%moderateof the tumor cells.overexpression.3+A strong complete membrane strongstaining is detected in >10%overexpression.of the tumor cells.

[0131] The above IHC staining patterns are routinely used in determining HER2-positive breast cancer. The terms HER2(+), HER2(++) and HER2(+++) used herein are equivalent to the terms HER2(1+), HER2(2+) and HER2(3+). A “low protein expression level” used in context of this invention corresponds to a 0 or 1+ score (“negative assessment” according to the table shown herein above), an “weak to moderate protein expression level” corresponds to a 2+ score (“weak to moderate overexpression”, see the table above) and a “high protein expression level” corresponds to a 3+ score (“strong overexpression”, see the table above). As described herein above in detail, the evaluation of the protein expression level (i.e. the scoring system as shown in the table) is based on results obtained by immunohistochemical methods. As a standard or routinely, the HER-2 status is, accordingly, performed by immunohistochemistry with one of two FDA-approved commercial kits available; namely the Dako Herceptest™ and the Ventana Pathway™. These are semi-quantitative assays which stratify expression levels into 0 (<20,000 receptors per cell, no expression visible by IHC staining), 1+(˜100,000 receptors per cell, partial membrane staining, <10% of cells overexpressing HER-2), 2+(˜500,000 receptors per cell, light to moderate complete membrane staining, >10% of cells overexpressing HER-2), and 3+(˜2,000,000 receptors per cell, strong complete membrane staining, >10% of cells overexpressing HER-2).

[0132] Alternatively, further methods for the evaluation of the protein expression level of HER2 may be used, e.g. Western Blots, ELISA-based detection systems and so on.

[0133] A HER2-positive cancer may also be diagnosed by assessing the gene amplification status of HER2. HER2-positive cancer is, accordingly, diagnosed if this assessment by ISH is positive. In accordance with this assessment, a HER2-positive cancer may, inter alia, relate to an average HER2 gene copy number higher than 4 copies of the HER2 gene per tumor cell (for those test systems without an internal centromere control probe) or to a HER2 / CEP17 ratio of >=2.0 (for those test systems using an internal chromosome 17 centromere control probe). In other words, the HER2-positive cancer may, inter alia, relate to a HER2 gene copy number greater than 4. The amplification level of the HER2 gene may easily be identified by in situ hybridization (ISH) like fluorescent in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and silver in situ hybridization (SISH). These methods are known to the skilled artisan. The principles of these methods can be deduced from standard text books. Commercial kits for the determination of the HER2 gene amplification status by in situ hybridization are available.

[0134] The below IHC staining patterns are recommended for determining HER2-positive gastric cancer (see Dako Herceptest package insert).of Hercep Test™ stained biopsies a cluster of at least 5 stained tumor cells is recommended. A cluster of at least 5 stained tumor cells consists of 5 connected HER2 stained tumor cells.TABLE 9Interpretation and scoring of HER2 immunohistochemical stainingHER2Over-Surgical Specimen-Biopsy Specimen-expressionScoreStaining PatternStaining PatternAssessment0 No reactivity No reactivity or Negativeor membranousno membranousreactivity in reactivity in any <10% of tumor cells(or <5 clustered)tumor cell1+Faint / barely perceptibleTumor cell cluster Negativemembranous (≥5 cells) with areactivity in ≥10% offaint / barely perceptible tumor cells, cells membranousare reactive only inreactivity irrespective part of their membraneof percentageof tumor cells stained2+Weak to moderate Tumor cell cluster Equivocalcomplete, basolateral (≥5 cells) with a weak or lateral membranousto moderate complete,reactivity in ≥10% basolateral or of tumor cellslateral membranousreactivity irrespective of percentageof tumor cells stained3+Strong complete, Tumor cell cluster Positivebasolateral or(≥5 cells) with a strong lateral membranous complete, basolateral orreactivitylateral membranous in ≥10% of tumor cellsreactivity irrespective of percentage of tumorcells stainedGuidelines based on Hofmann et al. (40).More refined IHC staining patterns for determining HER2-positive gastric cancer is as follows:HER2StainingOver-IntensitySurgical specimen-Biopsy specimen-expressionScorestaining patternstaining patternAssessment0 No reactivity or noNo reactivity or noNegativemembranous reactivitymembranous in <10% of tumour cellsreactivity in anytumour cell1+Faint / barely perceptibleTumour cell cluster (≥5Negativemembranous reactivitycells) with a faint / barelyin ≥10% of tumour cells;perceptible membranouscells are reactive only inreactivity irrespective ofpart of their membranepercentage of tumour cells stained2+Weak to moderateTumour cell cluster (≥5Equivocalcomplete, basolateral orcells) with a weak tolateral membranousmoderate complete,reactivity in ≥10% ofbasolateral or lateraltumour cellsmembranous reactivityirrespective of percentage oftumour cells stained3+Strong complete, Tumour cell cluster (≥5Positivebasolateralcells) with a strongor lateral membranouscomplete, basolateral orreactivity in ≥10% oflateral membranous tumour cellsreactivity irrespective of percentage oftumour cells stainedAs indicated above, the HER2 positive cancer to be treated in accordance with the present invention may be breast cancer, such early stage breast cancer. The term “early-stage breast cancer” as used herein refers to breast cancer that has not spread beyond the breast or the axillary lymph nodes. Such cancer can be generally treated with neoadjuvant or adjuvant therapy. The term “neoadjuvant therapy” as used herein refers to systemic therapy given prior to surgery. The term “adjuvant therapy” refers to systemic therapy given after surgery. In accordance with the above, treatment may be neoadjuvant or adjuvant therapy of early-stage breast cancer.

[0137] In accordance with the above, the sample to be assessed can be (obtained) from a patient with HER2-positive cancer as defined above. For example, the sample may be obtained from a tumorous tissue, (a) tumor(s) and, accordingly, is (a) tumor cell(s) or (a) tumor tissue(s) suspected of being HER2-positive tumour, like a breast tumor and the like. A person skilled in the art is in the position to identify such tumors and / or individuals / patients suffering from corresponding cancer using standard techniques known in the art and methods disclosed herein. Generally, said tumor cell or cancer cell may be obtained from any biological source / organism, particularly any biological source / organism, suffering from the above-mentioned cancer. In context of this invention particular useful cells are, preferably, human cells. These cells can be obtained from e.g. biopsies or from biological samples. The tumor / cancer / tumor cell / cancer cell is a solid tumor / cancer / tumor cell / cancer cell. In accordance with the above, the cancer / tumor cell may be a breast cancer / tumor cell or said sample comprises a cancer / tumor cell, such as a breast cancer / tumor cell. In line with the above, said tumor / cancer may be a breast tumor / cancer.

[0138] The modulator of the HER2 / neu (ErbB2) signaling pathway may be an inhibitor of HER2, for example, a HER dimerization / signaling inhibitor. The HER dimerization inhibitor may be a HER2 dimerization inhibitor. The HER dimerization inhibitor may inhibit HER heterodimerization or HER homodimerization. The HER dimerization inhibitor may be an anti-HER antibody. The term “antibody” herein is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity. Also human and humanized as well as CDR-grafted antibodies are comprised within the term “antibody”.

[0139] The HER antibody may bind to a HER receptor selected from the group consisting of EGFR, HER2 and HER3. Preferably, the antibody binds to HER2. The anti HER2 antibody may bind to domain II of HER2 extracellular domain. The antibody may bind to a junction between domains I, II and III of HER2 extracellular domain. The anti HER2 antibody may be Pertuzumab.

[0140] For the purposes herein, “Pertuzumab” and “rhuMAb 2C4”, which are used interchangeably, refer to an antibody comprising the variable light and variable heavy domains (amino acid sequences thereof shown in SEQ ID Nos. 5 and 6, respectively, as depicted in FIG. 2). The variable light and variable heavy domains of variant 574 / Pertuzumab are also shown in FIG. 2 (amino acid sequences thereof shown in SEQ ID Nos. 7 and 8, respectively, as depicted in FIG. 2). Where Pertuzumab is an intact antibody, it preferably comprises an IgG1 antibody; in one embodiment comprising the light chain amino acid sequence in it preferably comprises the light chain and heavy chain amino acid sequences, respectively, as shown in FIGS. 3A / 3B and 5A / 5B (FIG. 5A / 5B show the light chain and heavy chain amino acid sequences of a variant Pertuzumab). The heavy chain amino acid sequences of Pertuzumab as shown in FIG. 3B may optionally comprise an additional amino acid “K” at position 449 at the C-terminus. The antibody is optionally produced by recombinant Chinese Hamster Ovary (CHO) cells. The terms “Pertuzumab” and “rhuMAb 2C4” herein cover biosimilar versions of the drug with the United States Adopted Name (USAN) or International Nonproprietary Name (INN): Pertuzumab. Again, corresponding sequences are shown in FIGS. 2 to 5.

[0141] The modulator of the HER2 / neu (ErbB2) signaling pathway may be an inhibitor of HER shedding, for example a HER2 shedding inhibitor. The inhibitor of HER shedding may inhibit HER heterodimerization or HER homodimerization. Said inhibitor of HER shedding may be an anti-HER antibody.

[0142] The anti-HER antibody may bind to a HER receptor selected from the group consisting of EGFR, HER2 and HER3. Preferably, the antibody binds to HER2. The HER2 antibody may bind to sub-domain IV of the HER2 extracellular domain. Preferably, the HER2 antibody is Herceptin™ / Trastuzumab.

[0143] For the purposes herein, “Herceptin™” / “Trastuzumab” and “rhuMAb4D5-8”, which are used interchangeably, refer to an antibody comprising the variable light domains and variable heavy domains (amino acid sequences thereof are shown in FIG. 4, respectively; the domain is indicated by arrows). Where Trastuzumab is an intact antibody, it preferably comprises an IgG1 antibody; in one embodiment comprising the light chain amino and the heavy chain amino acid sequence as shown in FIG. 4. The antibody is optionally produced by Chinese Hamster Ovary (CHO) cells. The terms “Trastuzumab” and “rhuMAb4D5-8” herein cover biosimilar versions of the drug with the United States Adopted Name (USAN) or International Nonproprietary Name (INN): Trastuzumab.

[0144] The inhibitor of programmed death ligand 1 (PD-L1) may be an antibody specifically binding to PD-L1 (anti-PD-L1 antibody).

[0145] Exemplary anti-PD-L1 antibodies are disclosed in WO 2010 / 077634 which is incorporated herein in its entirety. Corresponding exemplary anti-PD-L1 antibodies to be used in accordance with the present invention are described below.

[0146] The anti-PD-L1 antibody may comprise a heavy chain variable region polypeptide comprising an HVR-H1, HVR-H2 and HVR-H3 sequence, wherein:(SEQ ID NO: 1)(a) the HVR-H1 sequence is GFTFSX1SWIEI;(SEQ ID NO: 2)(b) the HVR-H2 sequence is AWIX2PYGGSX3YYADSVKG;(SEQ ID NO: 3)(c) the HVR-H3 sequence is RHWPGGFDY;further wherein: X1 is D or G; X2 is S or L; X3 is T or S. X1 may be D; X2 may be S and X3 may be T.

[0147] The polypeptide may further comprise variable region heavy chain framework sequences juxtaposed between the HVRs according to the formula: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4). The framework sequences may be derived from human consensus framework sequences. The framework sequences may be VH subgroup III consensus framework. One or more of the framework sequences may be the following:(SEQ ID NO: 4)HC-FR1 is EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO: 5)HC-FR2 is WVRQAPGKGLEWV(SEQ ID NO: 6)HC-FR3 is RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR(SEQ ID NO: 7)HC-FR4 is WGQGTLVTVSA.

[0148] The heavy chain polypeptide may be in combination with a variable region light chain comprising an HVR-L1, HVR-L2 and HVR-L3, wherein:(a) the HVR-L1 sequence is(SEQ ID NOs: 8)RASQX4X5X6TX7X8A;(b) the HVR-L2 sequence is(SEQ ID NOs: 9)SASX9LX10S,;and(c) the HVR-L3 sequence is(SEQ ID NOs: 10)QQX11X12X13X14PX15T;further wherein: X4 is D or V; X5 is V or I; X6 is S or N; X7 is A or F; X8 is V or L; X9 is F or T; X10 is Y or A; X11 is Y, G, F, or S; X12 is L, Y, F or W; X13 is Y, N, A, T, G, F or I; X14 is H, V, P, T or I; X15 is A, W, R, P or T.

[0149] X4 may be D; X5 may be V; X6 may be S; X7 may be A; X8 may be V; X9 may be F; X10 may be Y; X11 may be Y; X12 may be L; X13 may be Y; X14 may be H; X15 may be A.

[0150] The polypeptide may further comprise variable region light chain framework sequences juxtaposed between the HVRs according to the formula: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). The framework sequences may be derived from human consensus framework sequences. The framework sequences may be VL kappa I consensus framework. One or more of the framework sequences may be the following:LC-FR1 is(SEQ ID NO: 11)DIQMTQSPSSLSASVGDRVTITC;LC-FR2 is(SEQ ID NO: 12)WYQQKPGKAPKLLIY;LC-FR3 is(SEQ ID NO: 13)GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC;LC-FR4 is(SEQ ID NO: 14)FGQGTKVEIKR.

[0151] The anti-PD-L1 antibody (or an antigen binding fragment thereof) may comprise a heavy chain and a light chain variable region sequence, wherein:(a) the heavy chain comprises an HVR-H1, HVR-H2and HVR-H3, wherein further:(SEQ ID NO: 1)(i) the HVR-H1 sequence is GFTFSX1SWIH;(SEQ ID NO: 2)(ii) the HVR-H2 sequence is AWIX2PYGGSX3YYADSVKG;(SEQ ID NO: 3)(iii) the HVR-H3 sequence is RHWPGGFDY, and(b) the light chain comprises an HVR-L1, HVR-L2and HVR-L3, wherein further:(SEQ ID NOs: 8)(iv) the HVR-L1 sequence is RASQX4X5X6TX7X8A;(SEQ ID NOs: 9)(v) the HVR-L2 sequence is SASX9LX10S;(SEQ ID NOs: 10)(vi) the HVR-L3 sequence is QQX11X12X13X14PX15T;wherein: X1 is D or G; X2 is S or L; X3 is T or S; X4 may be D or V; X5 may be V or I; X6 may be S or N; X7 may be A or F; X8 may be V or L; X9 may be F or T; X10 may be Y or A; X11 may be Y, G, F, or S; X12 may be L, Y, F or W; X13 may be Y, N, A, T, G, F or I; X14 may be H, V, P, T or I; X15 may be A, W, R, P or T.

[0153] X1 may be D; X2 may be S and X3 may be T. Furthermore, the positions may be as follows: X4=D, X5=V, X6=S, X7=A and X8=V, X9=F, and X10=Y, X11=Y, X12=L, X13=Y, X14=H and / or X15=A. Furthermore, the positions may be as follows: X1=D, X2=S and X3=T, X4=D, X5=V, X6=S, X7=A and X8=V, X9=F, and X10=Y, X11=Y, X12=L, X13=Y, X14=H and X15=A.

[0154] The antibody (an antigen binding fragment thereof) may further comprise

[0155] (a) variable region heavy chain framework sequences juxtaposed between the HVRs according to the formula: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and

[0156] (b) variable region light chain framework sequences juxtaposed between the HVRs according to the formula: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). The framework sequences may be derived from human consensus framework sequences.

[0157] The variable region heavy chain framework sequences may be VH subgroup III consensus framework. One or more of the framework sequences may be the following:(SEQ ID NO: 4)HC-FR1 is EVQLVESGGGLVQPGGSLRLSCAAS;(SEQ ID NO: 5)HC-FR2 is WVRQAPGKGLEWV;(SEQ ID NO: 6)HC-FR3 is RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR;(SEQ ID NO: 7)HC-FR4 is WGQGTLVTVSA.

[0158] The variable region light chain framework sequences may be VL kappa I consensus framework. One or more of the framework sequences may be the following:(SEQ ID NO: 11)LC-FR1 is DIQMTQSPSSLSASVGDRVTITC;(SEQ ID NO: 12)LC-FR2 is WYQQKPGKAPKLLIY;(SEQ ID NO: 13)LC-FR3 is GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC,;and;(SEQ ID NO: 14)LC-FR4 is FGQGTKVEIKR.

[0159] The antibody or antigen binding fragment thereof) may be or may comprise(a) the variable heavy chain framework sequencesare the following:(SEQ ID NO: 4)(i) HC-FR1 is EVQLVESGGGLVQPGGSLRLSCAAS;(SEQ ID NO: 5)(ii) HC-FR2 is WVRQAPGKGLEWV;(SEQ ID NO: 6)(iii) HC-FR3 is RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR;(SEQ ID NO: 7)(iv) HC-FR4 is WGQGTLVTVSA;;and(b) the variable light chain framework sequencesare the following:(SEQ ID NO: 11)(i) LC-FR1 is DIQMTQSPSSLSASVGDRVTITC;(SEQ ID NO: 12)(ii) LC-FR2 is WYQQKPGKAPKLLIY;(SEQ ID NO: 13)(iii) LC-FR3 is GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC;(SEQ ID NO: 14)(iv) LC-FR4 is FGQGTKVEIKR.

[0160] The antibody (or fragment thereof) may further comprise a human constant region. The constant region may selected from the group consisting of IgG1, IgG2, IgG3 and IgG4. The constant region may be IgG1. The antibody (or fragment thereof) may further comprise murine constant region. The constant region may be selected from the group consisting of IgG1, IgG2A, IgG2B and IgG3. The constant region may be IgG2A.

[0161] The antibody (or fragment thereof) may have reduced or minimal effector function. The minimal effector function may result from an effector-less Fc mutation. The effector-less Fc mutation may be N297A. The effector-less Fc mutation may be D265A / N297A. The minimal effector function may result from aglycosylation.

[0162] The antibody (or fragment thereof) may comprise a heavy chain and a light chain variable region sequence, wherein:

[0163] (a) the heavy chain comprises an HVR-H1, HVR-H2 and an HVR-H3, having at least 85% overall sequence identity to GFTFSDSWIH (SEQ ID NO:15), AWISPYGGSTYYADSVKG (SEQ ID NO:16) and RHWPGGFDY (SEQ ID NO:3), respectively, and

[0164] (b) the light chain comprises an HVR-L1, HVR-L2 and an HVR-L3, having at least 85% overall sequence identity to RASQDVSTAVA (SEQ ID NO:17), SASFLYS (SEQ ID NO:18) and QQYLYHPAT (SEQ ID NO:19), respectively.

[0165] The sequence identity may be at least 90%.

[0166] The antibody (or fragment thereof) may further comprise:

[0167] (a) variable region heavy chain (VH) framework sequences juxtaposed between the HVRs according to the formula: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and

[0168] (b) variable region light chain (VL) framework sequences juxtaposed between the HVRs according to the formula: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4).

[0169] The antibody (or fragment thereof) may further comprise a VH and VL framework region derived from a human consensus sequence. The VH framework sequence may be derived from a Kabat subgroup I, II, or III sequence. The VH framework sequence may be a Kabat subgroup III consensus framework sequence. The VH framework sequences may be the following:(SEQ ID NO: 4)HC-FR1 is EVQLVESGGGLVQPGGSLRLSCAAS;(SEQ ID NO: 5)HC-FR2 is WVRQAPGKGLEWV;(SEQ ID NO: 6)HC-FR3 is RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR;(SEQ ID NO: 7)HC-FR4 is WGQGTLVTVSA.

[0170] The VL framework sequence may be derived from a Kabat kappa I, II, III or IV subgroup sequence. The VL framework sequence may be a Kabat kappa I consensus framework sequence.

[0171] The VL framework sequences may be the following:(SEQ ID NO: 11)LC-FR1 is DIQMTQSPSSLSASVGDRVTITC;(SEQ ID NO: 12)LC-FR2 is WYQQKPGKAPKLLIY;(SEQ ID NO: 13)LC-FR3 is GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC;(SEQ ID NO: 14)LC-FR4 is FGQGTKVEIKR.

[0172] The antibody (or fragment thereof) may comprise a heavy chain and a light chain variable region sequence, wherein:(a) the heavy chain sequence has at least 85%sequence identity to the heavy chain sequence:(SEQ ID NO: 20)EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSA,and(b) the light chain sequence has at least 85%sequence identity to the light chain sequence:(SEQ ID NO: 21)DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR.

[0173] The sequence identity may be at least 90%.

[0174] The antibody (or fragment thereof) may comprise a heavy chain and light chain variable region sequence, wherein:(a) the heavy chain comprises the sequence:(SEQ ID NO: 20)EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSA,and(b) the light chain comprises the sequence:(SEQ ID NO: 21)DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR.

[0175] Moreover, the anti-PD-L1 antibody may be encoded by a nucleic acid. Accordingly, herein described is an isolated nucleic acid encoding the above polypeptide / antibody (or fragment thereof).

[0176] Provided herein is an isolated nucleic acid encoding a light chain or a heavy chain variable sequence of an anti-PD-L1 antibody or antigen binding fragment, wherein:

[0177] (a) the heavy chain further comprises and HVR-H1, HVR-H2 and an HVR-H3 sequence having at least 85% sequence identity to GFTFSDSWIH (SEQ ID NO:15), AWISPYGGSTYYADSVKG (SEQ ID NO:16) and RHWPGGFDY (SEQ ID NO:3), respectively, or

[0178] (b) the light chain further comprises an HVR-L1, HVR-L2 and an HVR-L3 sequence having at least 85% sequence identity to RASQDVSTAVA (SEQ ID NO:17), SASFLYS (SEQ ID NO:18) and QQYLYHPAT (SEQ ID NO:19), respectively.

[0179] The sequence identity may be 90%. The anti-PD-L1 antibody may further comprise a VL and a VH framework region derived from a human consensus sequence. The VH sequence may be derived from a Kabat subgroup I, II, or III sequence. The VL sequence may be derived from a Kabat kappa I, II, III or IV subgroup sequence. The anti-PD-L1 antibody may comprise a constant region derived from a murine antibody. The anti-PD-L1 antibody may comprise a constant region derived from a human antibody. The constant region may be IgG1. The antibody encoded by the nucleic acid may have reduced or minimal effector function. The minimal effector function may result from an effector-less Fc mutation. The effector-less Fc mutation may be N297A.

[0180] Further provided herein is a vector comprising the nucleic acid, a host cell comprising the vector. The host cell may be eukaryotic. The host cell may be mammalian. The host cell may be a Chinese Hamster Ovary (CHO) cell. The host cell may be prokaryotic. The host cell may be E. coli. Also provided herein is a process for making an anti-PD-L1 antibody comprising culturing the above host cell under conditions suitable for the expression of the vector encoding the anti-PD-L1 antibody or antigen binding fragment, and recovering the antibody or fragment.

[0181] The following describes in more detail the herein provided means and methods for treating a cancer and / or a cancer patient.

[0182] Herein contemplated is, accordingly, a pharmaceutical composition comprising a modulator of the HER2 / neu (ErbB2) signaling pathway (like Trastuzumab), and an inhibitor of programmed death ligand 1 (PD-L1) (like the anti-PD-L1 antibody described herein) for use in the treatment of cancer, whereby said cancer is determined to have a low or absent ER expression level and to have an increased expression level of programmed death ligand 1 (PD-L1) in comparison to a control. The cancer may be determined to have a decreased expression level of interferon-gamma (IFNγ) in comparison to the control. The pharmaceutical composition may further comprise a chemotherapeutic agent (like taxol or a taxol derivative, such as dodetaxel (Taxotere®)).

[0183] In accordance with the above, the present invention provides a method for treating cancer comprising administering an effective amount of a modulator of the HER2 / neu (ErbB2) signaling pathway, a chemotherapeutic agent and an inhibitor of programmed death ligand 1 (PD-L1) to a subject in need thereof. The cancer may be determined to have a decreased expression level of interferon-gamma (IFNγ) in comparison to the control.

[0184] Herein provided is a modulator of the HER2 / neu (ErbB2) signaling pathway, and an inhibitor of programmed death ligand 1 (PD-L1) for use in the treatment of cancer, whereby said cancer is determined to have a low or absent ER expression level and to have an increased expression level of programmed death ligand 1 (PD-L1) in comparison to a control. Moreover, herein provided is a modulator of the HER2 / neu (ErbB2) signaling pathway, an inhibitor of programmed death ligand 1 (PD-L1) and a chemotherapeutic agent (like taxol or a taxol derivative, such as dodetaxel (Taxotere®)) for use in the treatment of cancer, whereby said cancer is determined to have a low or absent ER expression level and to have an increased expression level of programmed death ligand 1 (PD-L1) in comparison to a control. The cancer may be determined to have a decreased expression level of interferon-gamma (IFNγ) in comparison to the control.

[0185] As discussed above, the present invention provides a method of treating a cancer in a cancer patient for whom therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated, the method comprising selecting a cancer patient whose cancer is determined to have a low or absent ER expression level and to have an increased expression level of programmed death ligand 1 (PD-L1) in comparison to a control, and administering to the patient an effective amount of a modulator of the HER2 / neu (ErbB2) signaling pathway, of a chemotherapeutic agent and of a programmed death ligand 1 (PD-L1) inhibitor. Likewise, the present invention provides a method of treating a cancer in a cancer patient who is undergoing therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent, the method comprising selecting a cancer patient whose cancer is determined to have a low or absent ER expression level and to have an increased expression level of programmed death ligand 1 (PD-L1) in comparison to a control, and administering to the patient an effective amount of a programmed death ligand 1 (PD-L1) inhibitor.

[0186] The explanations and definitions given herein above in relation to “cancer”, “cancer patient”, “PD-L1 inhibitor”, “PD-L1 inhibitor therapy”, “modulator of the HER2 / neu (ErbB2) signaling pathway”, “chemotherapeutic agent”, “low or absent ER expression level”“increased expression level of programmed death ligand 1 (PD-L1)”, “decreased expression level of interferon-gamma (IFN-γ) and the like apply, mutatis mutandis, in the context of the herein.

[0187] The terms “treatment”, “treating” and the like are used herein to generally mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of partially or completely curing a disease and / or adverse effect attributed to the disease. The term “treatment” as used herein covers any treatment of a disease in a patient and includes: (a) preventing a disease related in a patient which may be predisposed to the disease; (b) inhibiting the disease, i.e. arresting its development; or (c) relieving the disease, i.e. causing regression of the disease.

[0188] A “patient” for the purposes of the present invention includes both humans and other animals, particularly mammals, and other organisms. Thus, the methods are applicable to both human therapy and veterinary applications. Preferably, the patient is human.

[0189] The below explanations relate in more detail to the treatment / therapy of these patients / this patient group in accordance with the present invention.

[0190] The pharmaceutical composition will be formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the individual patient, the site of delivery of the pharmaceutical composition, the method of administration, the scheduling of administration, and other factors known to practitioners. The “effective amount” of the pharmaceutical composition for purposes herein is thus determined by such considerations.

[0191] The skilled person knows that the effective amount of one of the herein described PD-L1 inhibitor(s), modulator(s) of the HER2 / neu (ErbB2) signaling pathway and chemotherapeutic agent(s) in a pharmaceutical composition administered to an individual will, inter alia, depend on the nature of the compound. For example, if said compound is a (poly)peptide or protein the total pharmaceutically effective amount of pharmaceutical composition administered parenterally per dose will be in the range of about 1 μg protein / kg / day to 10 mg protein / kg / day of patient body weight, although, as noted above, this will be subject to therapeutic discretion. More preferably, this dose is at least 0.01 mg protein / kg / day, and most preferably for humans between about 0.01 and 1 mg protein / kg / day.

[0192] The following administration may be employed in respect of Trastuzumab:

[0193] Posology and method of administration

[0194] HER2 testing is mandatory prior to initiation of therapy. Herceptin treatment should only be initiated by a physician experienced in the administration of cytotoxic chemotherapy.MBCThree-Weekly Schedule

[0195] The recommended initial loading dose is 8 mg / kg body weight. The recommended maintenance dose at three-weekly intervals is 6 mg / kg body weight, beginning three weeks after the loading dose.Weekly Schedule

[0196] The recommended initial loading dose of Herceptin is 4 mg / kg body weight. The recommended weekly maintenance dose of Herceptin is 2 mg / kg body weight, beginning one week after the loading dose.Administration in Combination with Paclitaxel or Docetaxel

[0197] In the pivotal trials (H0648g, M77001), paclitaxel or docetaxel was administered the day following the first dose of Herceptin (for dose, see the Summary of Product Characteristics for paclitaxel or docetaxel) and immediately after the subsequent doses of Herceptin if the preceding dose of Herceptin was well tolerated.Administration in Combination with an Aromatase Inhibitor

[0198] In the pivotal trial (B016216) Herceptin and anastrozole were administered from day 1. There were no restrictions on the relative timing of Herceptin and anastrozole at administration (for dose, see the Summary of Product Characteristics for anastrozole or other aromatase inhibitors).EBCThree-Weekly and Weekly Schedule

[0199] As a three-weekly regimen the recommended initial loading dose of Herceptin is 8 mg / kg body weight. The recommended maintenance dose of Herceptin at three-weekly intervals is 6 mg / kg body weight, beginning three weeks after the loading dose.

[0200] As a weekly regimen (initial loading dose of 4 mg / kg followed by 2 mg / kg every week) concomitantly with paclitaxel following chemotherapy with doxorubicin and cyclophosphamide.

[0201] (See section 5.1 for chemotherapy combination dosing).MGCThree-Weekly Schedule

[0202] The recommended initial loading dose is 8 mg / kg body weight. The recommended maintenance dose at three-weekly intervals is 6 mg / kg body weight, beginning three weeks after the loading dose.Breast Cancer (MBC and EBC) and Gastric Cancer (MGC)Duration of Treatment

[0203] Patients with MBC or MGC should be treated with Herceptin until progression of disease. Patients with EBC should be treated with Herceptin for 1 year or until disease recurrence, whatever occurs first.Dose Reduction

[0204] No reductions in the dose of Herceptin were made during clinical trials. Patients may continue therapy during periods of reversible, chemotherapy-induced myelosuppression but they should be monitored carefully for complications of neutropenia during this time. Refer to the Summary of Product Characteristics for paclitaxel, docetaxel or aromatase inhibitor for information on dose reduction or delays.Missed Doses

[0205] If the patient misses a dose of Herceptin by one week or less, then the usual maintenance dose (weekly regimen: 2 mg / kg; three-weekly regimen: 6 mg / kg) should be given as soon as possible. Do not wait until the next planned cycle. Subsequent maintenance doses (weekly regimen: 2 mg / kg; three-weekly regimen: 6 mg / kg respectively) should then be given according to the previous schedule.

[0206] If the patient misses a dose of Herceptin by more than one week, a re-loading dose of Herceptin should be given over approximately 90 minutes (weekly regimen: 4 mg / kg; three-weekly regimen: 8 mg / kg). Subsequent Herceptin maintenance doses (weekly regimen: 2 mg / kg; three-weekly regimen 6 mg / kg respectively) should then be given (weekly regimen: every week; three-weekly regimen every 3 weeks) from that point.Special Patient Populations

[0207] Clinical data show that the disposition of Herceptin is not altered based on age or serum creatinine In clinical trials, elderly patients did not receive reduced doses of Herceptin. Dedicated pharmacokinetic studies in the elderly and those with renal or hepatic impairment have not been carried out. However, in a population pharmacokinetic analysis, age and renal impairment were not shown to affect trastuzumab disposition.Method of Administration

[0208] Herceptin loading dose should be administered as a 90-minute intravenous infusion. Do not administer as an intravenous push or bolus. Herceptin intravenous infusion should be administered by a health-care provider prepared to manage anaphylaxis and an emergency kit should be available. Patients should be observed for at least six hours after the start of the first infusion and for two hours after the start of the subsequent infusions for symptoms like fever and chills or other infusion-related symptoms (see sections 4.4 and 4.8). Interruption or slowing the rate of the infusion may help control such symptoms. The infusion may be resumed when symptoms abate.

[0209] If the initial loading dose was well tolerated, the subsequent doses can be administered as a 30-minute infusion. Pharmaceutical compositions of the invention may be administered parenterally.

[0210] Pharmaceutical compositions of the invention preferably comprise a pharmaceutically acceptable carrier. By “pharmaceutically acceptable carrier” is meant a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The term “parenteral” as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion. The administration of the herein provided compositions may, inter alia, comprise an administration twice daily, every day, every other day, every third day, every fourth day, every fifth day, once a week, once every second week, once every third week, once every month, etc.

[0211] The pharmaceutical composition is also suitably administered by sustained release systems. Suitable examples of sustained-release compositions include semi-permeable polymer matrices in the form of shaped articles, e.g., films, or mirocapsules. Sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman, U. et al., Biopolymers 22:547-556 (1983)), poly (2-hydroxyethyl methacrylate) (R. Langer et al., J. Biomed. Mater. Res. 15:167-277 (1981), and R. Langer, Chem. Tech. 12:98-105 (1982)), ethylene vinyl acetate (R. Langer et al., Id.) or poly-D-(−)-3-hydroxybutyric acid (EP 133,988). Sustained release pharmaceutical composition also include liposomally entrapped compound. Liposomes containing the pharmaceutical composition are prepared by methods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. (USA) 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. percent cholesterol, the selected proportion being adjusted for the optimal therapy.

[0212] For parenteral administration, the pharmaceutical composition is formulated generally by mixing it at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.

[0213] Generally, the formulations are prepared by contacting the components of the pharmaceutical composition uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product is shaped into the desired formulation. Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Non aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes. The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) (poly)peptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, manose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and / or nonionic surfactants such as polysorbates, poloxamers, or PEG.

[0214] The components of the pharmaceutical composition to be used for therapeutic administration must be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). Therapeutic components of the pharmaceutical composition generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0215] The components of the pharmaceutical composition ordinarily will be stored in unit or multi-dose containers, for example, sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution. As an example of a lyophilized formulation, 10-ml vials are filled with 5 ml of sterile-filtered 1% (w / v) aqueous solution, and the resulting mixture is lyophilized. The infusion solution is prepared by reconstituting the lyophilized compound(s) using bacteriostatic Water-for-Injection.

[0216] The herein provided treatment of cancer comprising a modulator of the HER2 / neu (ErbB2) signaling pathway, an inhibitor of programmed death ligand 1 (PD-L1) and a chemotherapeutic agent (like taxol or a taxol derivative, such as dodetaxel (Taxotere®)) may be performed by way of the simultaneous, sequential or separate administration of the individual components of said treatment. For example, one or more of the modulator(s) of the HER2 / neu (ErbB2) signaling pathway as defined herein (like Trastuzumab) may be administered simultaneously with one or more of the herein defined inhibitor(s) of programmed death ligand 1 (PD-L1) (like the herein provided and described anti-PD-L1 antibodies). Also, sequential administration of the modulator(s) of the HER2 / neu (ErbB2) signaling pathway as defined herein (like Trastuzumab) may be administered simultaneously with one or more of the herein defined inhibitor(s) of programmed death ligand 1 (PD-L1) (like the herein provided and described anti-PD-L1 antibodies) to be used in accordance with the present invention is envisaged herein. The herein defined modulators of the HER2 / neu (ErbB2) signaling pathway as defined herein (like Trastuzumab) and the one or more of the herein defined inhibitor of programmed death ligand 1 (PD-L1) (like the herein provided and described anti-PD-L1 antibodies) may also be administered separately. For example, one or more of the modulator(s) of the HER2 / neu (ErbB2) signaling pathway as defined herein (like Trastuzumab) may be administered in a first step followed by administration in a second step with one or more of the inhibitor(s) of programmed death ligand 1 (PD-L1) (like the herein provided and described anti-PD-L1 antibodies) and vice versa. Likewise, the chemotherapeutic agent may be administered simultaneously, sequentially or separately. Any combination of simultaneous, sequential or separate administration of the modulator(s) of the HER2 / neu (ErbB2) signaling pathway, inhibitor(s) of programmed death ligand 1 (PD-L1) and chemotherapeutic agent(s) (like taxol or a taxol derivative, such as dodetaxel (Taxotere®)) is envisaged herein.

[0217] The herein provided treatment of cancer comprising a modulator of the HER2 / neu (ErbB2) signaling pathway, an inhibitor of programmed death ligand 1 (PD-L1) and a chemotherapeutic agent (like taxol or a taxol derivative, such as dodetaxel (Taxotere®)) can be applied as a sole therapy. It may, however, also be applied with one or more additional therapies (i.e. in a further cotherapy with), for example, conventional therapies like surgery, radiotherapy and / or one or more additional chemotherapeutic agents.

[0218] Surgery may comprise the step of partial or complete tumour resection, prior to, during or after the administration of the herein provided cancer treatment comprising a modulator of the HER2 / neu (ErbB2) signaling pathway, an inhibitor of programmed death ligand 1 (PD-L1) and a chemotherapeutic agent (like taxol or a taxol derivative, such as dodetaxel (Taxotere®)). The herein provided modulator of the HER2 / neu (ErbB2) signaling pathway, inhibitor of programmed death ligand 1 (PD-L1) and chemotherapeutic agent (like taxol or a taxol derivative, such as dodetaxel (Taxotere®)) may be administered in a neoadjuvant or adjuvant setting (in particular neoadjuvant or adjuvant treatment of cancer).

[0219] The modulator of the HER2 / neu (ErbB2) signaling pathway, the chemotherapeutic agent and the inhibitor of programmed death ligand 1 (PD-L1) can be administered in a neoadjuvant setting. The modulator of the HER2 / neu (ErbB2) signaling pathway, the chemotherapeutic agent and the inhibitor of programmed death ligand 1 (PD-L1) can be administered in an adjuvant setting or in a metastatic setting.

[0220] Accordingly, the herein provided modulator of the HER2 / neu (ErbB2) signaling pathway, an inhibitor of programmed death ligand 1 (PD-L1) and a chemotherapeutic agent (like taxol or a taxol derivative, such as dodetaxel (Taxotere®)) may be administered to a patient in need of such a treatment during or after a surgical intervention / resection of the cancerous tissue. Therefore, the present invention is useful in neoadjuvant therapy, i.e. the treatment with the herein provided therapy given to a patient / patient group in need thereof prior to surgery. It is also useful in adjuvant therapy (i.e. after surgery).

[0221] The chemotherapeutic agent to be used herein is preferably a taxane (the term “taxol” is used interchangeably herein with “taxane”) or a taxane derivate (taxol derivative), like dodetaxel (Taxotere®) or paclitaxel. The use of dodetaxel / (Taxotere®) is particularly preferred herein.

[0222] The (additional) chemotherapeutic agent(s) may be one or more of the following exemplary, non-limiting, drugs or agents:

[0223] Cisplatin, Vinorelbin, Carboplatin, Paclitaxel, Gemcitabin, Docetaxel, Bevacizumab, Pemetrexed, Etoposid, Irinotecan, Ifosfamid, Topotecan,

[0224] (an) anti-angiogenic agent(s) like a VEGF blocker (such as bevacizumab / Avastin or sutent (sunitinib malate-SU-11248)), linomide, inhibitors of integrin αvβ3 function, angiostatin, razoxin, thalidomide, and including vascular targeting agents (for example combretastatin phosphate or N-acetylcolchinol-O-phosphate));

[0225] (an) cytostatic agent(s) such as antioestrogens (for example tamoxifen, toremifene, raloxifene, droloxifene, iodoxyfene), progestogens (for example megestrol acetate), aromatase inhibitors (for example anastrozole, letrazole, vorazole, exemestane), antiprogestogens, antiandrogens (for example flutamide, nilutamide, bicalutamide, cyproterone acetate), LHRH agonists and antagonists (for example goserelin acetate, luprolide), inhibitors of testosterone 5α-dihydroreductase (for example finasteride), anti-invasion agents (for example

[0226] metalloproteinase inhibitors like marimastat and inhibitors of urokinase plasminogen activator receptor function) and inhibitors of growth factor function, (such growth factors include for example platelet derived growth factor and hepatocyte growth factor such inhibitors include growth factor antibodies, growth factor receptor antibodies, tyrosine kinase inhibitors and serine / threonine kinase inhibitors);

[0227] biological response modifiers (for example interferon); (an) anti-metabolite agent(s) (for example gemcitabine); (an) anti-hormonal compound(s) such as (an) anti-estrogen(s); antibodies (for example edrecolomab); adjuvant (anti-) hormonal therapy / therapies (i.e. therapy with (an) adjuvant (anti-) hormone drug(s), such as tamoxifen; gene therapy approaches (like antisense therapies); and / or immunotherapy approaches.

[0228] The chemotherapy may also (additionally) include the use of one or more of antiproliferative / antineoplastic drugs and combinations thereof, as used in medical oncology, such as (an) tyrosine kinase inhibitor(s), (a) raf inhibitor(s), (a) ras inhibitor(s), (a) dual tyrosine kinase inhibitor(s), taxol, (an) taxane(s) (like paclitaxel or docetaxel), (an) anthracycline(s), like doxorubicin or epirubicin, aromatase inhibitors (such as anastrozole or letrozole) and / or vinorelbine; cyclophosphamide, methotrexate or fluorouracil (which is also known as 5-FU) can be used in such cotherapy individually or in form of a cotherapy comprising these three drugs (“CMF therapy”), optionally in combination with any of the other herein provided additional therapies. Particular examples of chemotherapeutic agents for use with a combination treatment of the present invention are pemetrexed, raltitrexed, etoposide, vinorelbine, paclitaxel, docetaxel, cisplatin, oxaliplatin, carboplatin, gemcitabine, irinotecan (CPT-1 1), 5-fluorouracil (5-FU, (including capecitabine)), doxorubicin, cyclophosphamide, temozolomide, hydroxyurea, (iii) antiproliferative / antineoplastic drugs and combinations thereof, as used in medical oncology, such as antimetabolites (for example antifolates like methotrexate,

[0229] fluoropyrimidines like 5-fluorouracil, purine and adenosine analogues, cytosine arabinoside); antitumour antibiotics (for example anthracyclines like doxorubicin, daunomycin, epirubicin and idarubicin, mitomycin-C, dactinomycin, mithramycin); platinum derivatives (for example cisplatin, carboplatin); alkylating agents (for example nitrogen mustard, melphalan, chlorambucil, busulphan, cyclophosphamide, ifosfamide, nitrosoureas, thiotepa); antimitotic agents (for example vinca alkaloids like vincristine and taxoids like taxol, taxotere); topoisomerase inhibitors (for example epipodophyllotoxins like etoposide and teniposide, amsacrine, topotecan, and also irinotecan); also enzymes (for example asparaginase); and thymidylate synthase inhibitors (for example raltitrexed); and additional types of chemotherapeutic agents.

[0230] Inhibitors / Modulators / chemotherapeutic agents for use in accordance with the present invention are described herein and refer generally to known and / or commercially available Inhibitors / Modulators / chemotherapeutic. However, the use of inhibitors yet to be generated or known compounds to be tested for their inhibiting activity is envisaged in context of the present invention.

[0231] In a further aspect, the present invention relates to the use of (a) nucleic acid(s) or antibody (antibodies) capable of detecting the expression level of ER, PD-L1 and, optionally, IFNγ for determining a patient's need for PD-L1 inhibitor cotherapy in combination with a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent. The respective explanations of said terms have been given above and apply here mutatis mutandis.

[0232] Preferably, the nucleic acid (e.g. oligonucleotide(s)) is (are) about 15 to 100 nucleotides in length. A person skilled in the art is, based on his general knowledge and the teaching provided herein, easily in the position to identify and / or prepare (a) an oligo- or polynucleotide capable of detecting the expression level of ER, PD-L1 and, optionally, IFNγ. In particular these nucleic acid(s) (e.g. oligo- or polynucleotides) may be used as probe(s) in the methods described herein, for example in the measurement of the expression level. A skilled person will know, for example, computer programs which may be useful for the identification of corresponding probes to be used herein. For example, a nucleic acid encoding estrogen receptor (or a part of the nucleic acid) (e.g. SEQ ID NO: 38), a nucleic acid encoding PD-L1 (or a part of the nucleic acid) (e.g. SEQ ID NO: 42) and, optionally, a nucleic acid encoding IFNγ (or a part of the nucleic acid) (e.g. SEQ ID NO: 44 may be used in this context for identifying specific probes for detecting the expression level of ER, PD-L1 and IFNγ, respectively. Exemplary nucleic acid sequences encoding ER, PD-L1 and IFNγ are available on corresponding databases, such as the NCBI database (world wide web at ncbi.nlm.nih.gov / sites / entrez).

[0233] Furthermore, a composition is provided herein which is a diagnostic composition further comprising, optionally, means for detection / determining / evaluating the expression level of ER, PD-L1 and IFNγ. Such means for detection, are, for example, the above-described nucleotides and / or antibodies. Accordingly, the present invention relates to such means (e.g. such nucleotides and / or antibodies) for the preparation of a diagnostic composition for determining a patient in need of a PD-L1 inhibitor cotherapy.

[0234] In an alternative aspect, the present invention relates to such means for detection (e.g the above-described nucleic acids and / or antibodies and / or the “binding molecules” described below in context of the kit to be used in accordance with the present invention) for use in determining a patient in need of a PD-L1 inhibitor cotherapy. Preferably, the present invention relates to (an) antibody / antibodies for use in determining a patient in need of a PD-L1 inhibitor cotherapy.

[0235] Furthermore, the present invention also relates to a kit useful for carrying out the herein provided methods, the kit comprising (a) nucleic acid or (an) antibody capable of detecting the expression level of ER, PD-L1 and, optionally, IFNγ. Also envisaged herein is the use of the herein described kit for carrying out the herein provided methods. Said kit useful for carrying out the methods and uses described herein may comprise oligonucleotides or polynucleotides capable of determining the expression level of ER, PD-L1 and, optionally, IFNγ. For example, said kit may comprise (a) compound(s) required for specifically measuring the expression level of ER, PD-L1 and, optionally, IFNγ. Moreover, the present invention also relates to the use of (a) compound(s) required for specifically measuring the expression level of ER, PD-L1 and, optionally, IFNγ, for the preparation of a kit for carrying out the methods or uses of this invention. On the basis of the teaching of this invention, the skilled person knows which compound(s) is (are) required for specifically measuring the expression level of ER, PD-L1 and, optionally, IFNγ. For example, such compound(s) may be (a) “binding molecule(s)”. Particularly, such compound(s) may be (a) (nucleotide) probe(s), (a) primer(s) (pair(s)), (an) antibody(ies) and / or (an) aptamer(s) specific for a (gene) product of the ER gene / coding sequence, PD-L1 gene / coding sequence and, optionally, IFNγ / coding sequence. The kit (to be prepared in context) of this invention may be a diagnostic kit.

[0236] The kit (to be prepared in context) of this invention or the methods and uses of the invention may further comprise or be provided with (an) instruction manual(s). For example, said instruction manual(s) may guide the skilled person (how) to determine the (reference / control) expression level of ER, PD-L1 and, optionally, IFNγ. or (how) to determine a patient's need of PD-L1 inhibitor therapy. Particularly, said instruction manual(s) may comprise guidance to use or apply the herein provided methods or uses. The kit (to be prepared in context) of this invention may further comprise substances / chemicals and / or equipment suitable / required for carrying out the methods and uses of this invention. For example, such substances / chemicals and / or equipment are solvents, diluents and / or buffers for stabilizing and / or storing (a) compound(s) required for specifically measuring the expression level of ER, PD-L1 and, optionally, IFNγ.Definitions

[0237] As used herein, the terms “comprising” and “including” or grammatical variants thereof are to be taken as specifying the stated features, integers, steps or components but do not preclude the addition of one or more additional features, integers, steps, components or groups thereof. This term encompasses the terms “consisting of” and “consisting essentially of” Thus, the terms “comprising” / “including” / “having” mean that any further component (or likewise features, integers, steps and the like) can be present.

[0238] The term “consisting of” means that no further component (or likewise features, integers, steps and the like) can be present.

[0239] The term “consisting essentially of” or grammatical variants thereof when used herein are to be taken as specifying the stated features, integers, steps or components but do not preclude the addition of one or more additional features, integers, steps, components or groups thereof but only if the additional features, integers, steps, components or groups thereof do not materially alter the basic and novel characteristics of the claimed composition, device or method. Thus, the term “consisting essentially of” means that specific further components (or likewise features, integers, steps and the like) can be present, namely those not materially affecting the essential characteristics of the composition, device or method. In other words, the term “consisting essentially of” (which can be interchangeably used herein with the term “comprising substantially”), allows the presence of other components in the composition, device or method in addition to the mandatory components (or likewise features, integers, steps and the like), provided that the essential characteristics of the device or method are not materially affected by the presence of other components.

[0240] The term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, biological and biophysical arts.

[0241] As used herein, the term “isolated” refers to a composition that has been removed from its in-vivo location (e.g. aquatic organism or moss). Preferably the isolated compositions of the present invention are substantially free from other substances (e.g., other proteins that do not comprise anti-adhesive effects) that are present in their in-vivo location (i.e. purified or semi-purified).

[0242] As used herein the term “about” refers to +10%.ITEMS

[0243] The present invention also relates to the following items:

[0244] 1. A method of determining the need of a cancer patient for a PD-L1 inhibitor cotherapy,

[0245] (i) wherein therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated for the patient or (ii) wherein the patient is undergoing therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent, said method comprising the steps of

[0246] a) measuring in vitro in a sample from said patient the expression level of Estrogen receptor (ER) and of programmed death ligand 1 (PD-L1),

[0247] b) determining a patient as being in need of a PD-L1 inhibitor cotherapy if a low or absent ER expression level and an expression level of programmed death ligand 1 (PD-L1) that is increased in comparison to a control is measured in step (a).

[0248] 2. A method of treating a cancer in a cancer patient for whom therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated, the method comprising selecting a cancer patient whose cancer is determined to have a low or absent ER expression level and to have an increased expression level of programmed death ligand 1 (PD-L1) in comparison to a control, and administering to the patient an effective amount of a modulator of the HER2 / neu (ErbB2) signaling pathway, of a chemotherapeutic agent and of a programmed death ligand 1 (PD-L1) inhibitor.

[0249] 3. A method of treating a cancer in a cancer patient who is undergoing therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent, the method comprising selecting a cancer patient whose cancer is determined to have a low or absent ER expression level and to have an increased expression level of programmed death ligand 1 (PD-L1) in comparison to a control, and administering to the patient an effective amount of a programmed death ligand 1 (PD-L1) inhibitor.

[0250] 4. A pharmaceutical composition comprising a modulator of the HER2 / neu (ErbB2) signaling pathway, and an inhibitor of programmed death ligand 1 (PD-L1) for use in the treatment of cancer, whereby said cancer is determined to have a low or absent ER expression level and to have an increased expression level of programmed death ligand 1 (PD-L1) in comparison to a control.

[0251] 5. The pharmaceutical composition for use in the treatment of cancer of item 4, further comprising a chemotherapeutic agent.

[0252] 6. The method of any one of items 1 to 3, further comprising measuring in vitro in a sample from said patient the expression level of interferon-gamma (IFNγ) and determining a patient as being in need of a PD-L1 inhibitor cotherapy if an expression level of interferon-gamma (IFNγ) that is decreased in comparison to a control is measured.

[0253] 7. The method of any one of items 1, 2, 3 and 6; or the pharmaceutical composition of item 4 and 5, wherein the ER expression level is ER(−).

[0254] 8. The method of any one of items 1, 2, 3, 6 and 7; or the pharmaceutical composition of any one of item 4, 5 and 7, wherein said modulator of the HER2 / neu (ErbB2) signaling pathway is the HER2 antibody Herceptin / Trastuzumab.

[0255] 9. The method of any one of items 1, 2, 3, 6, 7 and 8; or the pharmaceutical composition of any one of items 5, 7 and 8, wherein said chemotherapeutic agent is taxol or a taxol derivative.

[0256] 10. The method of any one of items 1, 2, 3, 6, 7 and 8 to 9; or the pharmaceutical composition of any one of items 4, 5 and 7 to 9, wherein said inhibitor of programmed death ligand 1 (PD-L1) is an antibody specifically binding to PD-L1 (anti-PD-L1 antibody).

[0257] 11. The method of any one of items 1, 2, 3, and 6 to 10; or the pharmaceutical composition of any one of items 4, 5 and 7 to 10, wherein said cancer is a solid cancer.

[0258] 12. The method of item 11; or the pharmaceutical composition of item 11, wherein said solid cancer is breast cancer or gastric cancer

[0259] 13. The method of any one of items 1, 2, 3, and 6 to 12; or the pharmaceutical composition of any one of items 4, 5 and 7 to 12, wherein the expression level of PD-L1 is the mRNA expression level.

[0260] 14. Use of a nucleic acid or antibody capable of detecting the expression level of ER, PD-L1 and, optionally, IFNγ for determining a patient's need for PD-L1 inhibitor cotherapy in combination with a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent.

[0261] 15. The method of any one of items 1, 2, 3 and 6 to 14; or the pharmaceutical composition of any one of items 4, 5 and 7 to 14, wherein said modulator of the HER2 / neu (ErbB2) signaling pathway, said chemotherapeutic agent and said inhibitor of programmed death ligand 1 (PD-L1) are to be administered in a neoadjuvant setting.

[0262] The present invention is further described by reference to the following non-limiting figures and examples. Unless otherwise indicated, established methods of recombinant gene technology were used as described, for example, in Sambrook, Russell “Molecular Cloning, A Laboratory Manual”, Cold Spring Harbor Laboratory, N.Y. (2001) which is incorporated herein by reference in its entirety.BRIEF DESCRIPTION OF THE DRAWINGS

[0263] FIG. 1 provides a schematic of the HER2 protein structure, and amino acid sequences for Domains I-IV, respectively) of the extracellular domain thereof (SEQ ID NOS. 22-25, respectively, in order of appearance).

[0264] FIGS. 2A and 2B depict alignments of the amino acid sequences of the variable light (VL) (FIG. 2A) and variable heavy (VH) (FIG. 2B) domains of murine monoclonal antibody 2C4 (SEQ ID Nos. 26 and 27, respectively); VL and VH domains of variant 574 / Pertuzumab (SEQ ID Nos. 28 and 29, respectively), and human VL and VH consensus frameworks (hum κ1, light kappa subgroup I; humIII, heavy subgroup III) (SEQ ID Nos. 30 and 31, respectively). Asterisks identify differences between variable domains of Pertuzumab and murine monoclonal antibody 2C4 or between variable domains of Pertuzumab and the human framework. Complementarity Determining Regions (CDRs) are in brackets.

[0265] FIGS. 3A and 3B show the amino acid sequences of Pertuzumab light chain (FIG. 3A; SEQ ID NO: 32) and heavy chain (FIG. 3B; SEQ ID NO: 33). CDRs are shown in bold. Calculated molecular mass of the light chain and heavy chain are 23,526.22 Da and 49,216.56 Da (cysteines in reduced form). The carbohydrate moiety is attached to Asn 299 of the heavy chain.

[0266] FIGS. 4A and 4B show the amino acid sequences of Trastuzumab light chain (FIG. 4A; SEQ ID NO: 34) and heavy chain (FIG. 4B; SEQ ID NO: 35), respectively. Boundaries of the variable light and variable heavy domains are indicated by arrows.

[0267] FIGS. 5A and 5B depict a variant Pertuzumab light chain sequence (FIG. 5A; SEQ ID NO: 36) and a variant Pertuzumab heavy chain sequence (FIG. 5B; SEQ ID NO: 37), respectively.

[0268] FIG. 6: FIGS. 6A and 6B show known mRNA transcripts and position of the relevant AFFYMETRIX probe set target regions for gene CD274. Exons are shown as grey bold rectangles, junction regions are indicated by thin horizontal lines. Probe sets with their sequence mapped against mRNA sequences are shown as black bold rectangles. Provided coordinates are genomic coordinates on chromosome 9.

[0269] FIG. 7: FIGS. 7A and 7B show known mRNA transcripts and position of the relevant AFFYMETRIX probe set target regions for gene IFNG. Exons are shown as grey bold rectangles, junction regions are indicated by thin horizontal lines. Probe sets with their sequence mapped against mRNA sequences are shown as black bold rectangles. Provided coordinates are genomic coordinates on chromosome 12.

[0270] FIG. 8 shows the distribution of the expression of genes IFNG and CD274 in the samples of ER- and ER-30 populations. Symbol types correspond to the final pCR status (solid: pCR achieved, open—pCR not achieved).

[0271] FIG. 9A is a box plot of expression of gene CD274 for ER− responders (pCR=YES) and nonresponders (pCR=NO). On the right the histograms of expression for both categories are provided. FIG. 9B shows a distribution of t-test statistics (HO hypothesis of no difference). The vertical mark indicates the actual value found in the involved sample. The area of the shaded regions corresponds to the alpha level.

[0272] FIG. 10A is a box plot of expression of gene IFNG for ER− responders (pCR=YES) and nonresponders (pCR=NO). On the right the histograms of expression for both categories are provided. FIG. 10B shows the distribution of t-test statistics (HO hypothesis of no difference). The vertical mark indicates the actual value found in the involved sample. The area of the shaded regions corresponds to the alpha level.

[0273] FIG. 11A is a box plot of expression of gene CD274 for ER+ responders (pCR=YES) and nonresponders (pCR=NO). On the right the histograms of expression for both categories are provided. FIG. 11B shows a distribution of t-test statistics (HO hypothesis of no difference). The vertical mark indicates the actual value found in the involved sample. The area of the shaded regions corresponds to the alpha level.

[0274] FIG. 12A is a box plot of expression of gene IFNG for ER+ responders (pCR=YES) and nonresponders (pCR=NO). On the right the histograms of expression for both categories are provided. FIG. 12B shows a distribution of t-test statistics (HO hypothesis of no difference). The vertical mark indicates the actual value found in the involved sample. The area of the shaded regions corresponds to the alpha level.

[0275] FIG. 13 is the receiver operating characteristic of the final logistic regression model for ER-population. Positive level is taken to be the positive response status (pCR=YES).

[0276] FIG. 14 is a LIFT curve of the final logistic regression model for ER− population. Positive level is taken to be the positive response status (pCR=YES). The Y-axis displays the ratio of how rich the portion of the population is in the chosen response level (upper curve corresponds to pCR=YES) compared to the rate of that response level as a whole.

[0277] FIG. 15 is an example of predicted clinical response status for ER− population. Shown is predicted profile of response as controlled by patient age, Cancer type, pN status, and expression of both genes involved. The actual predicted pCR probability (which is equal to 0.443) is given for NO LABC patient around 60 y. old and with expression in both genes around median values.

[0278] FIG. 16 is the receiver operating characteristic of the final logistic regression model for ER+ population. Positive level is taken to be the positive response status (pCR=YES).

[0279] FIG. 17 shows the distribution of age of ER− patients.

[0280] FIG. 18 shows the distribution of age of ER+ patients.

[0281] FIG. 19 shows a decision tree view on expression of IFNG and CD274 genes predicting clinical response in ER patients. The first two splits required to explain pCR are the ones wrt to IFNG and CD274.EXAMPLES

[0282] The Example illustrates the invention.Example 1: Cancer Patients Undergoing HER2 Targeted Therapy and Chemotherapy Benefit from PD-L1 Inhibitor Cotherapy, if the Expression Level of ER is Low or Absent (ER Negative) and if PD-L1 Expression Level is Increased

[0283] Estimation of gene expression was performed with the help of R Bioconductor package ‘affy’, R version 2.15.0. All exploratory analyses and predictive models were made using SAS JMP ver. 10.0

[0284] 48 HER2+, ER+ and 39 HER2+, ER− breast cancer biopsies were obtained from NeoSphere clinical trial. The samples had been taken at diagnosis from patients afterwards treated with Docetaxel and Trastuzumab in a neo-adjuvant setting. The distribution of main clinical covariates at base line, as well as of clinical response (as assessed at the surgery) in the involved population is as follows:ER Negative Samples:Patient Age (see FIG. 17)Quantiles100.0%maximum72 99.5%72 97.5%71.55 90.0%64 75.0%quartile54 50.0%median50.5 25.0%quartile44.25 10.0%39 2.5%34.675 0.5%34 0.0%minimum34LevelCountProbCancer TypeIBC 20.04167LABC220.45833OPERABLE240.50000Total481.00000pT (pathologic staging of Tumor)T2180.37500T3150.31250T4150.31250Total481.00000pN (pathologic staging of nodes)N0120.25000N1360.75000Total481.00000G (Grade)G1 10.02083G2150.31250G3160.33333NA160.33333Total481.00000ER Positive Samples:Patient Age (see FIG. 18)Quantiles100.0%maximum74 99.5%74 97.5%74 90.0%65 75.0%quartile57 50.0%median50 25.0%quartile43 10.0%40 2.5%32 0.5%32 0.0%minimum32LevelCountProbCancer TypeIBC 50.12821LABC 80.20513OPERABLE260.66667Total391.00000pTT2150.38462T3160.41026T4 80.20513Total391.00000pNN0110.28205N1280.71795Total391.00000GG2130.33333G3100.25641NA160.41026Total391.00000Contingency Analysis of pathological complete response (pCR) By estrogen receptor status (ER)Count Row %pCR = NOpCR = YESER = ER−2756.252143.7548ER = ER+3384.62615.3839602787Gene Expression ProfilingThe tumor biopsy samples were profiled for gene expression on AFFYMETRIX HG-U133Plus 2

[0287] whole Human Genome microarray platform. Roche HighPure RNA extraction, NuGen amplification and standard AFFYMETRIX hybridization and scanning protocols were used. All array scans passed standard AFFYMETRIX QC.

[0288] Robust Multiarray algorithm (RMA) was used for preprocessing of raw signals (Irizarry et al, 2003. available at ncbi.nlm.nih.gov / pubmed / 12925520). All probe sets available for the genes of interest were retrieved as reported below. For gene CD274, when several probe sets were available to represent this gene, the probe set with the probe set with the highest average expression value (defined as an arithmetical average of expression of a given probe set) was selected to represent the gene:

[0289] CD274 (PDL1)

[0290] 223834_at selected for PDL1

[0291] 227458_at

[0292] The selected probe set corresponds to the last exon / 3′UTR of the gene and captures all known RefSEq mRNAs (see FIGS. 6A and 6B)

[0293] IFNG

[0294] 210354_at

[0295] This probe set also represents the last exon / 3′UTR of the gene and captures all known RefSEq mRNAs (see FIGS. 7A and 7B)

[0296] FIG. 8 shows joint distribution of the expression of the above genes in the samples of both ER− and ER− populations. Symbol types correspond to the final pCR status (solid: pCRachieved, open—pCR not achieved).

[0297] More details on distribution of CD274 and IFNG expression across ER and pCR strata can be found in Appendix I.

[0298] For every ER subpopulation, a logistic regression model was constructed that relates expression of the selected genes with clinical response adjusted for patient age, cancer type, and nodal status:Response∼Patient.Age+Cancer.Type+pN+CD⁢274+IFNG1. ER− Population.

[0299] Summarized model output is given below. Odds ratios are (OR) provided per unit change of biomarker value. As the expression values are given on log 2 scale, one unit change would correspond to 2-fold overexpression. For details see Appendix.ER− populationTermOR (95% CI)LR test p-valueCD2745.2 (1.5; 26.7)0.008IFNG0.30 (0.10; 0.74)0.007Patient Age0.24Cancer Type0.91pN0.87

[0300] The final model for predicting probability for a particular patient to respond to the treatment includes expression of CD274 and IFNG and looks like:p⁡(p⁢C⁢R)=-3.7⁢3⁢7+1.607*CD⁢274-1.069*IFNG2. ER+ Population.

[0301] Summarized model output is given below. Odds ratios are (OR) provided per unit change of biomarker value. As the expression values are given on log 2 scale, one unit change would correspond to 2-fold overexpression. For details see Appendix.ER+ populationTermOR (95% CI)LR test p-valueCD2740.93IFNG0.23Patient Age0.34Cancer Type0.39pN0.92

[0302] The role of PDL1 expression is evident in ER− subpopulation of HER2+ breast cancer patients that underwent combinational treatment with Trastuzumab and chemotherapy in the neoadjuvant setting. Namely, overexpression of PDL1 at diagnosis corresponds to a lower rate of response to neoadjuvant therapy (i.e. a lower rate of response to combinational treatment with Trastuzumab and chemotherapy). This holds irrespective of patient age, cancer type, or lymph node status. A baseline assessment of gene expression of either of the two biomarkers, PDL1 and INFG, respectively, allows to identify if a patient is likely to experience a greater benefit if a PDL-1 targeted therapy is added to Trastuzumab and chemotherapy.

[0303] The following relates to a cut-off value allowing determining a patient as being in need of a PD-L1 inhibitor cotherapy in accordance with the present invention.

[0304] If a gene expression analysis gives a result for IFNG expression higher or equal to 4.8 no combination treatment (HER2-targeted and PDL1-targeted) is recommended and no further PDL1 assessment would be necessary. If a gene expression analysis gives a result for IFNG lower than 4.8 a parallel assessment of PDL-1 is necessary. If PDL-1 gene expression analysis then gives a result of higher or equal to 5.3 a combination treatment (HER2-targeted and PDL1-targeted) is recommended (see FIG. 19).APPENDIX IER− SubpopulationOneway Analysis of CD274 Expression by pCR ER=ERneg (See FIG. 9a)t Test

[0306] YES-NO

[0307] Assuming unequal variancesDifference−0.32948t Ratio−1.94171Std Err Dif0.16969DF45.11513Upper CL Dif0.01226Prob > |t|0.0584Lower CL Dif−0.67122Prob > t0.9708Confidence0.95Prob < t0.0292*

[0308] The results are also shown in FIG. 9B.Oneway Analysis of IFNG Expression by pCR ER=ERneg

[0309] The results are shown in FIG. 10A.

[0310] t Test

[0311] YES-NO

[0312] Assuming unequal variancesDifference0.58405t Ratio2.044225Std Err Dif0.28571DF30.21429Upper CL Dif1.16737Prob > |t|0.0497*Lower CL Dif0.00073Prob > t0.0249*Confidence0.95Prob < t0.9751

[0313] The results are shown in FIG. 10B.ER+ SubpopulationOneway Analysis of CD274 Expression by pCR ER=ERpos

[0314] The results are shown in FIG. 11A.

[0315] t Test

[0316] YES-NO

[0317] Assuming unequal variancesDifference0.25169t Ratio0.898709Std Err Dif0.28006DF6.542171Upper CL Dif0.92345Prob > |t|0.4007Lower CL Dif−0.42006Prob > t0.2003Confidence0.95Prob < t0.7997

[0318] The results are shown in FIG. 11B.Oneway Analysis of IFNG Expression by pCR ER=ERpos

[0319] The results are shown in FIG. 12A.

[0320] t Test

[0321] YES-NO

[0322] Assuming unequal variancesDifference0.5931t Ratio1.501336Std Err Dif0.3951DF7.109044Upper CL Dif1.5244Prob > |t|0.1763Lower CL Dif−0.3382Prob > t0.0882Confidence0.95Prob < t0.9118

[0323] The results are shown in FIG. 12B.APPENDIX II

[0324] Nominal Logistic Fit for pCR ER=ERneg

[0325] Converged in Gradient, 5 iterationsWhole Model TestModel−LogLikelihoodDFChiSquareProb > ChiSqDifference6.784783613.569570.0348*Full26.110299Reduced32.895082RSquare (U)0.2063AICc69.0206BIC79.319Observations 48(or Sum Wgts)MeasureTraining DefinitionEntropy RSquare0.2063 1-Loglike(model) / Loglike(0)Generalized RSquare0.3301 (1 − (L(0) / L(model)){circumflex over ( )}(2 / n)) / (1 − L(0){circumflex over ( )}(2 / n))Mean −Log p0.5440 Σ−Log(ρ[j]) / nRMSE0.4278 √Σ(y[j]−ρ[j])2 / nMean Abs Dev0.3665 Σ |y[j]−ρ[j]| / nMisclassification Rate0.2292 Σ (ρ[j]≠ρMax) / nN48 nLack of FitSourceDF−LogLikelihoodChiSquareLack Of Fit4126.11029952.2206Saturated470.000000Prob > ChiSqFitted626.1102990.1125Parameter EstimatesProb >TermEstimateStd ErrorChiSquareChiSqLower 95%Upper 95%Intercept−5.96882554.16326952.060.1517−15.1153291.70408281Patient Age0.049062380.04250451.330.2484−0.03240340.13829525Cancer Type[IBC]−0.09430231.09822890.010.9316−2.54079772.23824618Cancer Type[LABC]−0.15149450.65444240.050.8169−1.50512691.21757158pN[N0]0.081576360.49795740.030.8699−0.89866221.09707358CD274 Expression1.649792220.71947625.260.0218*0.395338333.2836052IFNG Expression−1.18829780.51220235.380.0203*−2.3323039−0.2889168For log odds of NO / YESEffect Likelihood Ratio TestsSourceNparmDFL-R ChiSquareProb > ChiSqPatient Age111.385744460.2391Cancer Type220.197810330.9058pN110.026907040.8697CD274 Expression117.098004330.0077*IFNG Expression117.153877230.0075*Odds RatioFor pCR odds of NO versus YESTests and confidence intervals on odds ratios are likelihood ratio based.Unit Odds RatiosPer unit change in regressorTermOdds RatioLower 95%Upper 95%ReciprocalPatient Age1.0502860.9681161.1483150.9521217CD2745.2058981.48488626.671760.1920898ExpressionIFNG0.304740.0970720.7490743.2814908ExpressionOdds Ratios for Cancer TypeOddsProb >LowerUpperLevel1 / Level2RatioChisq95%95%LABCIBC0.94441250.97220.028298935.902054OPER-IBC1.4050870.84710.035747968.159191ABLEOPER-LABC1.48778950.65680.25187699.0216463ABLEIBCLABC1.05885930.97220.027853635.337072IBCOPER-0.71169970.84710.014671527.973694ABLELABCOPER-0.67213810.65680.11084453.9701934ABLEOdds Ratios for pNProb >LowerUpperLevel1 / Level2Odds RatioChisq95%95%N1N00.84946150.86970.11145366.033483N0N11.17721650.86970.16574178.9723459Receiver Operating Characteristic(see FIG. 13)Using pCR=‘YES’ to be the positive levelAUC0.79718Confusion Matrix

[0335] Actual

[0336] PredictedTrainingNOYESNO225YES615Lift Curve (see FIG. 14)pCRNO

[0339] YES

[0340] Prediction Profiler (see FIG. 15)

[0341] Nominal Logistic Fit for pCR ER=ERpos

[0342] Converged in Gradient, 19 iterationsWhole Model TestModel−LogLikelihoodDFChiSquareProb > ChiSqDifference2.40059764.8011930.5696Full14.343001Reduced16.743598RSquare (U)0.1434AICc46.2989BIC54.3309Observations (or Sum Wgts)39MeasureTraining DefinitionEntropy RSquare0.1434 1-Loglike(model) / Loglike(0)Generalized RSquare0.2010 (1 − (L(0) / L(model)){circumflex over ( )}(2 / n)) / (1 − L(0){circumflex over ( )}(2 / n))Mean −Log p0.3678 Σ−Log(ρ[j]) / nRMSE0.3462 √Σ(y[j]−ρ[j])2 / nMean Abs Dev0.2351 Σ |y[j]−ρ[j]| / nMisclassification Rate0.1795 Σ (ρ[j]≠ρMax) / nN39 nLack Of FitSourceDF−LogLikelihoodChiSquareLack Of Fit3214.34300128.686Saturated380.000000Prob > ChiSqFitted614.3430010.6351Parameter EstimatesProb >TermEstimateStd ErrorChiSquareChiSqLower 95%Upper 95%InterceptUnstable7.203069093597.51070.000.9984−7043.78847058.1945Patient Age0.05781490.06281120.850.3573−0.05604830.19608254CancerUnstable12.00925137195.01390.000.9987−14089.95914113.9773Type[IBC]CancerUnstable−6.58646833597.5070.000.9985−7057.57067044.39766Type[LABC]pN[N0]−0.05428690.55729040.010.9224−1.16983781.117206CD2740.084852710.98591640.010.9314−1.87046982.14104768ExpressionIFNG−0.73346780.61918171.400.2362−2.04769030.45985303ExpressionFor log odds of NO / YESEffect Likelihood Ratio TestsSourceNparmDFL-R ChiSquareProb > ChiSqPatient Age110.925887320.3359Cancer Type221.891402120.3884pN110.009464440.9225CD274 Expression110.007422130.9313IFNG Expression111.456939450.2274Odds RatiosFor pCR odds of NO versus YESTests and confidence intervals on odds ratios are likelihood ratio based.Unit Odds RatiosPer unit change in regressorOddsLowerUpperTermRatio95%95%ReciprocalPatient Age1.0595190.9454931.2166270.9438246CD274 Expression1.0885570.1540518.5083470.9186476IFNG Expression0.4802410.1290331.5838412.0822891Odds Ratios for Cancer TypeLevel1 / Level2Odds RatioProb > ChisqLower 95%Upper 95%LABCIBC8.3942e−90.212805.1523961OPERABLEIBC2.6876e−80.4499020.868673OPERABLELABC3.20171120.31930.299926236.429388IBCLABC1191292510.21280.1940845.IBCOPERABLE372079930.44990.0479187.LABCOPERABLE0.3123330.31930.02745043.3341535Odds Ratios for pNOddsProb >LowerUpperLevel1 / Level2RatioChisq95%95%N1N01.11468720.92250.107055110.377869N0N10.89711260.92250.09635899.3409878Receiver Operating Characteristic(see FIG. 16)Using pCR=‘YES’ to be the positive levelAUC0.77273Confusion MatrixActualPredictedTrainingNOYESNO321YES60The present invention refers to the following nucleotide and amino acid sequences: The sequences provided herein are, inter alia, available in the NCBI database and disclosed in WO 2010 / 077634 and can be retrieved from world wide web at ncbi.nlm.nih.gov / sites / entrez?db=gene; Theses sequences also relate to annotated and modified sequences. The present invention also provides techniques and methods wherein homologous sequences, and variants of the concise sequences provided herein are used.

[0354] SEQ ID NOS: 1-21 define the anti-PD-L1 antibody to be used in accordance with the present invention. SEQ ID NOS: 1-21 are shown in the sequence listing.

[0355] SEQ ID No. 22 to 37 show sequences of amino acid sequences for Domains I-IV of the HER2 protein (SEQ ID NO. 22-25, see also FIG. 1) and sequences of anti-HER2-antibodies. (SEQ ID NOS: 26 to 37; see also FIGS. 2A, 2B, 3A, 3B, 4A, 4B, 5A, and 5B).SEQ ID No. 26:Amino acid sequence of the variable

[0357] light (Vr) (FIG. 2A) domain of murine monoclonal antibody 2C4 (SEQ ID NOS: 26 and 27, respectively) as shown in FIGS. 2A and 2B.SEQ ID No. 27:Amino acid sequence of the variable heavy (VH) (FIG. 2B) domain of murine monoclonal antibody 2C4 as shown in FIGS. 2A and 2B.SEQ ID No. 28:Amino acid sequence of the variable light (VL) (FIG. 2A) domain of variant 574 / Pertuzumab as shown in FIGS. 2A and 2B.SEQ ID No. 29:Amino acid sequence of the variable heavy (VH) (FIG. 2B) domain of variant 574 / Pertuzumab as shown in FIGS. 2A and 2B.SEQ ID No. 30:human VL consensus frameworks (hum κ1, light kappa subgroup I; humIII, heavy subgroup III) as shown in FIGS. 2A and 2B.SEQ ID No. 31:human VH consensus frameworks (hum κ1, light kappa subgroup I; humIII, heavy subgroup III) as shown in FIGS. 2A and 2B.SEQ ID No. 32:Amino acid sequences of Pertuzumab light chain as shown in FIG. 3A.SEQ ID No. 33:Amino acid sequences of Pertuzumab heavy chain as shown in FIG. 3B.SEQ ID No. 34:Amino acid sequence of Trastuzumab light chain domain as shown in FIG. 4A Boundaries of the variable light domain are indicated by arrows.SEQ ID No. 35:Amino acid sequence of Trastuzumab heavy chain as shown in FIG. 4B. Boundaries of the variable heavy domain are indicated by arrows.SEQ ID No. 36:Amino acid sequence of variant Pertuzumab light chain sequence (FIG. 5A).SEQ ID No. 37:Amino acid sequence of variant Pertuzumab heavy chain sequence (FIG. 5B).SEQ ID NO. 38:Nucleotide sequence encoding Homo sapiens Progesterone Receptor (PR)NCBI Reference Sequence: NC_000011.9>gi|224589802:c101000544-100900355 Homo sapiens chromosome 11, GRCh37.p10 Primary AssemblySEQ ID No. 39:Amino acid sequence of Homo sapiens Progesterone Receptor (PR)PRGR_HUMAN Length: 933 Dec. 7, 2012 15:10 Type: P Check: 6067.SEQ ID NO. 40:Nucleotide sequence encoding Homo sapiens Estrogen Receptor (ER)(NM_000125.3)SEQ ID NO. 41:Nucleotide sequence encoding Homo sapiens Estrogen Receptor (ER)NCBI Reference Sequence: NC_000006.11>gi|224589818:152011631-152424409 Homo sapiens chromosome 6, GRCh37.p10 Primary AssemblySEQ ID No. 42:Amino acid sequence of Homo sapiens Estrogen Receptor (ER)>ENST00000206249_6SEQ ID No. 43:Nucleotide sequence encoding Homo sapiens programmed death ligand 1 (PD-L1)NCBI Reference Sequence: NC_000009.11>gi|224589821:5450503-5470567 Homo sapiens chromosome 9, GRCh37.p10 Primary AssemblySEQ ID NO. 44Nucleotide sequence encoding Homo sapiens programmed death ligand 1 (PD-L1) (CD274), transcript variant 1, mRNANCBI Reference Sequence: NM_014143.3>gi|292658763|ref|NM_014143.31 Homo sapiens CD274 molecule (CD274), transcript variant 1,mRNASEQ ID No. 45:Amino acid sequence of Homo sapiens programmed death ligand 1 (PD-L1) (programmed cell death 1 ligand 1 isoform a precursor [Homo sapiens])NCBI Reference Sequence: NP_054862.1>gi|76615341ref|NP_054862.1| programmed cell death 1 ligand 1 isoform a precursor [Homo sapiens]SEQ ID No. 46:Nucleotide sequence encoding Homo sapiens programmed death ligand 1 (PD-L1) (CD274), transcript variant 2, mRNANCBI Reference Sequence: NM_001267706.1>gi|3909796381ref|NM_001267706.11 Homo sapiens CD274 molecule (CD274), transcript variant 2, mRNASEQ ID No. 47:Amino acid sequence of Homo sapiens programmed death ligand 1 (PD-L1) (programmed cell death 1 ligand 1 isoform b precursor [Homo sapiens])NCBI Reference Sequence: NP_001254635.1>gi|3909796391ref|NP_001254635.11 programmed cell death 1 ligand 1 isoform b precursor [Homo sapiens]SEQ ID No. 48:Nucleotide sequence encoding Homo sapiens programmed death ligand 1 (PD-L1) (Homo sapiens CD274 molecule (CD274), transcript variant 3, non-coding RNA)NCBI Reference Sequence: NR_052005.1>gi|3909796401ref|NR_052005.1| Homo sapiens CD274 molecule (CD274), transcript variant 3, non-coding RNASEQ ID No. 49:Nucleotide sequence encoding Homo sapiens interferon gamma (Homo sapiens chromosome 12, GRCh37.p10 Primary Assembly)NCBI Reference Sequence: NC_000012.11>gi|224589803:c68553521-68548550 Homo sapiens chromosome 12, GRCh37.p10 Primary AssemblySEQ ID No. 50:Nucleotide sequence encoding Homo sapiens interferon gamma, mRNANCBI Reference Sequence: NM_000619.2

[0405] >gi|56786137|ref|NM_000619.21 Homo sapiens interferon, gamma (IFNG), mRNASEQ ID No. 51:Amino acid sequence of Homo sapiens interferon gamma, interferon gamma precursor [Homo sapiens]

[0407] NCBI Reference Sequence: NP_000610.2

[0408] >gi|56786138|ref|NP_000610.21 interferon gamma precursor [Homo sapiens]

[0409] All references cited herein are fully incorporated by reference. Having now fully described the invention, it will be understood by a person skilled in the art that the invention may be practiced within a wide and equivalent range of conditions, parameters and the like, without affecting the spirit or scope of the invention or any embodiment thereof.SEQUENCE LISTINGThe patent application contains a lengthy sequence listing. A copy of the sequence listing is available in electronic form from the USPTO web site (). An electronic copy of the sequence listing will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3).Sequence total quantity: 51 Current application number: US / 19 / 067,334 SEQ ID NO: 1 moltype = AA length = 10 FEATURE Location / Qualifiers REGION 1..10 note = Description of Artificial Sequence: Synthetic HVR-H1 peptide VARIANT 6 note = Asp or Gly source 1..10 mol_type = protein organism = synthetic construct SEQUENCE: 1 GFTFSXSWIH 10 SEQ ID NO: 2 moltype = AA length = 18 FEATURE Location / Qualifiers REGION 1..18 note = Description of Artificial Sequence: Synthetic HVR-H2 peptide VARIANT 4 note = Ser or Leu VARIANT 10 note = Thr or Ser source 1..18 mol_type = protein organism = synthetic construct SEQUENCE: 2 AWIXPYGGSX YYADSVKG 18 SEQ ID NO: 3 moltype = AA length = 9 FEATURE Location / Qualifiers REGION 1..9 note = Description of Artificial Sequence: Synthetic HVR-H3 peptide source 1..9 mol_type = protein organism = synthetic construct SEQUENCE: 3 RHWPGGFDY 9 SEQ ID NO: 4 moltype = AA length = 25 FEATURE Location / Qualifiers REGION 1..25 note = Description of Artificial Sequence: Synthetic HC-FR1 peptide source 1..25 mol_type = protein organism = synthetic construct SEQUENCE: 4 EVQLVESGGG LVQPGGSLRL SCAAS 25 SEQ ID NO: 5 moltype = AA length = 13 FEATURE Location / Qualifiers REGION 1..13 note = Description of Artificial Sequence: Synthetic HC-FR2 speptide source 1..13 mol_type = protein organism = synthetic construct SEQUENCE: 5 WVRQAPGKGL EWV 13 SEQ ID NO: 6 moltype = AA length = 32 FEATURE Location / Qualifiers REGION 1..32 note = Description of Artificial Sequence: Synthetic HC-FR3 polypeptide source 1..32 mol_type = protein organism = synthetic construct SEQUENCE: 6 RFTISADTSK NTAYLQMNSL RAEDTAVYYC AR 32 SEQ ID NO: 7 moltype = AA length = 11 FEATURE Location / Qualifiers REGION 1..11 note = Description of Artificial Sequence: Synthetic HC-FR4 peptide source 1..11 mol_type = protein organism = synthetic construct SEQUENCE: 7 WGQGTLVTVS A 11 SEQ ID NO: 8 moltype = AA length = 11 FEATURE Location / Qualifiers REGION 1..11 note = Description of Artificial Sequence: Synthetic HVR-L1 peptide VARIANT 5 note = Asp or Val VARIANT 6 note = Val or Ile VARIANT 7 note = Ser or Asn VARIANT 9 note = Ala or Phe VARIANT 10 note = Val or Leu source 1..11 mol_type = protein organism = synthetic construct SEQUENCE: 8 RASQXXXTXX A 11 SEQ ID NO: 9 moltype = AA length = 7 FEATURE Location / Qualifiers REGION 1..7 note = Description of Artificial Sequence: Synthetic HVR-L2 peptide VARIANT 4 note = Phe or Thr VARIANT 6 note = Tyr or Ala source 1..7 mol_type = protein organism = synthetic construct SEQUENCE: 9 SASXLXS 7 SEQ ID NO: 10 moltype = AA length = 9 FEATURE Location / Qualifiers REGION 1..9 note = Description of Artificial Sequence: Synthetic HVR-L3 peptide VARIANT 3 note = Tyr, Gly, Phe or Ser VARIANT 4 note = Leu, Tyr, Phe or Trp VARIANT 5 note = Tyr, Asn, Ala, Thr, Gly, Phe or Ile VARIANT 6 note = His, Val, Pro, Thr or Ile VARIANT 8 note = Ala, Trp, Arg, Pro or Thr source 1..9 mol_type = protein organism = synthetic construct SEQUENCE: 10 QQXXXXPXT 9 SEQ ID NO: 11 moltype = AA length = 23 FEATURE Location / Qualifiers REGION 1..23 note = Description of Artificial Sequence: Synthetic LC-FR1 peptide source 1..23 mol_type = protein organism = synthetic construct SEQUENCE: 11 DIQMTQSPSS LSASVGDRVT ITC 23 SEQ ID NO: 12 moltype = AA length = 15 FEATURE Location / Qualifiers REGION 1..15 note = Description of Artificial Sequence: Synthetic LC-FR2 peptide source 1..15 mol_type = protein organism = synthetic construct SEQUENCE: 12 WYQQKPGKAP KLLIY 15 SEQ ID NO: 13 moltype = AA length = 32 FEATURE Location / Qualifiers REGION 1..32 note = Description of Artificial Sequence: Synthetic LC-FR3 polypeptide source 1..32 mol_type = protein organism = synthetic construct SEQUENCE: 13 GVPSRFSGSG SGTDFTLTIS SLQPEDFATY YC 32 SEQ ID NO: 14 moltype = AA length = 11 FEATURE Location / Qualifiers REGION 1..11 note = Description of Artificial Sequence: Synthetic LC-FR4 peptide source 1..11 mol_type = protein organism = synthetic construct SEQUENCE: 14 FGQGTKVEIK R 11 SEQ ID NO: 15 moltype = AA length = 10 FEATURE Location / Qualifiers REGION 1..10 note = Description of Artificial Sequence: Synthetic heavy chain peptide source 1..10 mol_type = protein organism = synthetic construct SEQUENCE: 15 GFTFSDSWIH 10 SEQ ID NO: 16 moltype = AA length = 18 FEATURE Location / Qualifiers REGION 1..18 note = Description of Artificial Sequence: Synthetic heavy chain peptide source 1..18 mol_type = protein organism = synthetic construct SEQUENCE: 16 AWISPYGGST YYADSVKG 18 SEQ ID NO: 17 moltype = AA length = 11 FEATURE Location / Qualifiers REGION 1..11 note = Description of Artificial Sequence: Synthetic light chain peptide source 1..11 mol_type = protein organism = synthetic construct SEQUENCE: 17 RASQDVSTAV A 11 SEQ ID NO: 18 moltype = AA length = 7 FEATURE Location / Qualifiers REGION 1..7 note = Description of Artificial Sequence: Synthetic light chain peptide source 1..7 mol_type = protein organism = synthetic construct SEQUENCE: 18 SASFLYS 7 SEQ ID NO: 19 moltype = AA length = 9 FEATURE Location / Qualifiers REGION 1..9 note = Description of Artificial Sequence: Synthetic light chain peptide source 1..9 mol_type = protein organism = synthetic construct SEQUENCE: 19 QQYLYHPAT 9 SEQ ID NO: 20 moltype = AA length = 118 FEATURE Location / Qualifiers REGION 1..118 note = Description of Artificial Sequence: Synthetic heavy chain polypeptide source 1..118 mol_type = protein organism = synthetic construct SEQUENCE: 20 EVQLVESGGG LVQPGGSLRL SCAASGFTFS DSWIHWVRQA PGKGLEWVAW ISPYGGSTYY 60 ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARRH WPGGFDYWGQ GTLVTVSA 118 SEQ ID NO: 21 moltype = AA length = 108 FEATURE Location / Qualifiers REGION 1..108 note = Description of Artificial Sequence: Synthetic light chain polypeptide source 1..108 mol_type = protein organism = synthetic construct SEQUENCE: 21 DIQMTQSPSS LSASVGDRVT ITCRASQDVS TAVAWYQQKP GKAPKLLIYS ASFLYSGVPS 60 RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YLYHPATFGQ GTKVEIKR 108 SEQ ID NO: 22 moltype = AA length = 195 FEATURE Location / Qualifiers source 1..195 mol_type = protein organism = Homo sapiens SEQUENCE: 22 TQVCTGTDMK LRLPASPETH LDMLRHLYQG CQVVQGNLEL TYLPTNASLS FLQDIQEVQG 60 YVLIAHNQVR QVPLQRLRIV RGTQLFEDNY ALAVLDNGDP LNNTTPVTGA SPGGLRELQL 120 RSLTEILKGG VLIQRNPQLC YQDTILWKDI FHKNNQLALT LIDTNRSRAC HPCSPMCKGS 180 RCWGESSEDC QSLTR 195 SEQ ID NO: 23 moltype = AA length = 124 FEATURE Location / Qualifiers source 1..124 mol_type = protein organism = Homo sapiens SEQUENCE: 23 TVCAGGCARC KGPLPTDCCH EQCAAGCTGP KHSDCLACLH FNHSGICELH CPALVTYNTD 60 TFESMPNPEG RYTFGASCVT ACPYNYLSTD VGSCTLVCPL HNQEVTAEDG TQRCEKCSKP 120 CARV 124 SEQ ID NO: 24 moltype = AA length = 169 FEATURE Location / Qualifiers source 1..169 mol_type = protein organism = Homo sapiens SEQUENCE: 24 CYGLGMEHLR EVRAVTSANI QEFAGCKKIF GSLAFLPESF DGDPASNTAP LQPEQLQVFE 60 TLEEITGYLY ISAWPDSLPD LSVFQNLQVI RGRILHNGAY SLTLQGLGIS WLGLRSLREL 120 GSGLALIHHN THLCFVHTVP WDQLFRNPHQ ALLHTANRPE DECVGEGLA 169 SEQ ID NO: 25 moltype = AA length = 142 FEATURE Location / Qualifiers source 1..142 mol_type = protein organism = Homo sapiens SEQUENCE: 25 CHQLCARGHC WGPGPTQCVN CSQFLRGQEC VEECRVLQGL PREYVNARHC LPCHPECQPQ 60 NGSVTCFGPE ADQCVACAHY KDPPFCVARC PSGVKPDLSY MPIWKFPDEE GACQPCPINC 120 THSCVDLDDK GCPAEQRASP LT 142 SEQ ID NO: 26 moltype = AA length = 107 FEATURE Location / Qualifiers source 1..107 mol_type = protein organism = Mus musculus SEQUENCE: 26 DTVMTQSHKI MSTSVGDRVS ITCKASQDVS IGVAWYQQRP GQSPKLLIYS ASYRYTGVPD 60 RFTGSGSGTD FTFTISSVQA EDLAVYYCQQ YYIYPYTFGG GTKLEIK 107 SEQ ID NO: 27 moltype = AA length = 119 FEATURE Location / Qualifiers source 1..119 mol_type = protein organism = Mus musculus SEQUENCE: 27 EVQLQQSGPE LVKPGTSVKI SCKASGFTFT DYTMDWVKQS HGKSLEWIGD VNPNSGGSIY 60 NQRFKGKASL TVDRSSRIVY MELRSLTFED TAVYYCARNL GPSFYFDYWG QGTTLTVSS 119 SEQ ID NO: 28 moltype = AA length = 107 FEATURE Location / Qualifiers REGION 1..107 note = Description of Artificial Sequence: Synthetic humanized 574 Variable Light Chain source 1..107 mol_type = protein organism = synthetic construct SEQUENCE: 28 DIQMTQSPSS LSASVGDRVT ITCKASQDVS IGVAWYQQKP GKAPKLLIYS ASYRYTGVPS 60 RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YYIYPYTFGQ GTKVEIK 107 SEQ ID NO: 29 moltype = AA length = 119 FEATURE Location / Qualifiers REGION 1..119 note = Description of Artificial Sequence: Synthetic humanized 574 Variable Heavy Chain source 1..119 mol_type = protein organism = synthetic construct SEQUENCE: 29 EVQLVESGGG LVQPGGSLRL SCAASGFTFT DYTMDWVRQA PGKGLEWVAD VNPNSGGSIY 60 NQRFKGRFTL SVDRSKNTLY LQMNSLRAED TAVYYCARNL GPSFYFDYWG QGTLVTVSS 119 SEQ ID NO: 30 moltype = AA length = 107 FEATURE Location / Qualifiers REGION 1..107 note = Description of Artificial Sequence: Synthetic consensus framework Hum kappa1 Variable Light Chain source 1..107 mol_type = protein organism = synthetic construct SEQUENCE: 30 DIQMTQSPSS LSASVGDRVT ITCRASQSIS NYLAWYQQKP GKAPKLLIYA ASSLESGVPS 60 RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YNSLPWTFGQ GTKVEIK 107 SEQ ID NO: 31 moltype = AA length = 119 FEATURE Location / Qualifiers REGION 1..119 note = Description of Artificial Sequence: Synthetic consensus framework Hum kappa1 Variable Heavy Chain source 1..119 mol_type = protein organism = synthetic construct SEQUENCE: 31 EVQLVESGGG LVQPGGSLRL SCAASGFTFS SYAMSWVRQA PGKGLEWVAV ISGDGGSTYY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARGR VGYSLYDYWG QGTLVTVSS 119 SEQ ID NO: 32 moltype = AA length = 214 FEATURE Location / Qualifiers REGION 1..214 note = Description of Artificial Sequence: Synthetic Pertuzumab light chain source 1..214 mol_type = protein organism = synthetic construct SEQUENCE: 32 DIQMTQSPSS LSASVGDRVT ITCKASQDVS IGVAWYQQKP GKAPKLLIYS ASYRYTGVPS 60 RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YYIYPYTFGQ GTKVEIKRTV AAPSVFIFPP 120 SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT 180 LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 214 SEQ ID NO: 33 moltype = AA length = 448 FEATURE Location / Qualifiers REGION 1..448 note = Description of Artificial Sequence: Synthetic Pertuzumab heavy chain source 1..448 mol_type = protein organism = synthetic construct SEQUENCE: 33 EVQLVESGGG LVQPGGSLRL SCAASGFTFT DYTMDWVRQA PGKGLEWVAD VNPNSGGSIY 60 NQRFKGRFTL SVDRSKNTLY LQMNSLRAED TAVYYCARNL GPSFYFDYWG QGTLVTVSSA 120 STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG 180 LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKKVEPK SCDKTHTCPP CPAPELLGGP 240 SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS 300 TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSREEM 360 TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ 420 QGNVFSCSVM HEALHNHYTQ KSLSLSPG 448 SEQ ID NO: 34 moltype = AA length = 214 FEATURE Location / Qualifiers REGION 1..214 note = Description of Artificial Sequence: Synthetic Trastuzumab light chain source 1..214 mol_type = protein organism = synthetic construct SEQUENCE: 34 DIQMTQSPSS LSASVGDRVT ITCRASQDVN TAVAWYQQKP GKAPKLLIYS ASFLYSGVPS 60 RFSGSRSGTD FTLTISSLQP EDFATYYCQQ HYTTPPTFGQ GTKVEIKRTV AAPSVFIFPP 120 SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT 180 LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 214 SEQ ID NO: 35 moltype = AA length = 449 FEATURE Location / Qualifiers REGION 1..449 note = Description of Artificial Sequence: Synthetic Trastuzumab heavy chain source 1..449 mol_type = protein organism = synthetic construct SEQUENCE: 35 EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR IYPTNGYTRY 60 ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRWG GDGFYAMDYW GQGTLVTVSS 120 ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS 180 GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG 240 PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN 300 STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE 360 MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 420 QQGNVFSCSV MHEALHNHYT QKSLSLSPG 449 SEQ ID NO: 36 moltype = AA length = 217 FEATURE Location / Qualifiers REGION 1..217 note = Description of Artificial Sequence: Synthetic variant Pertuzumab light chain sequence source 1..217 mol_type = protein organism = synthetic construct SEQUENCE: 36 VHSDIQMTQS PSSLSASVGD RVTITCKASQ DVSIGVAWYQ QKPGKAPKLL IYSASYRYTG 60 VPSRFSGSGS GTDFTLTISS LQPEDFATYY CQQYYIYPYT FGQGTKVEIK RTVAAPSVFI 120 FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS 180 TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC 217 SEQ ID NO: 37 moltype = AA length = 449 FEATURE Location / Qualifiers REGION 1..449 note = Description of Artificial Sequence: Synthetic variant Pertuzumab heavy chain sequence source 1..449 mol_type = protein organism = synthetic construct SEQUENCE: 37 EVQLVESGGG LVQPGGSLRL SCAASGFTFT DYTMDWVRQA PGKGLEWVAD VNPNSGGSIY 60 NQRFKGRFTL SVDRSKNTLY LQMNSLRAED TAVYYCARNL GPSFYFDYWG QGTLVTVSSA 120 STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG 180 LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKKVEPK SCDKTHTCPP CPAPELLGGP 240 SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS 300 TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSREEM 360 TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ 420 QGNVFSCSVM HEALHNHYTQ KSLSLSPGK 449 SEQ ID NO: 38 moltype = DNA length = 100190 FEATURE Location / Qualifiers source 1..100190 mol_type = unassigned DNA organism = Homo sapiens SEQUENCE: 38 agtccacagc tgtcactaat cggggtaagc cttgttgtat ttgtgcgtgt gggtggcatt 60 ctcaatgaga actagcttca cttgtcattt gagtgaaatc tacaacccga ggcggctagt 120 gctcccgcac tactgggatc tgagatcttc ggagatgact gtcgcccgca gtacggagcc 180 agcagaagtc cgacccttcc tgggaatggg ctgtaccgag aggtccgact agccccaggg 240 ttttagtgag ggggcagtgg aactcagcga gggactgaga gcttcacagc atgcacgagt 300 ttgatgccag agaaaaagtc gggagataaa ggagccgcgt gtcactaaat tgccgtcgca 360 gccgcagcca ctcaagtgcc ggacttgtga gtactctgcg tctccagtcc tcggacagaa 420 gttggagaac tctcttggag aactccccga gttaggagac gagatctcct aacaattact 480 actttttctt gcgctcccca cttgccgctc gctgggacaa acgacagcca cagttcccct 540 gacgacagga tggaggccaa gggcaggagc tgaccagcgc cgccctcccc cgcccccgac 600 ccaggaggtg gagatccctc cggtccagcc acattcaaca cccactttct cctccctctg 660 cccctatatt cccgaaaccc cctcctcctt cccttttccc tcctcctgga gacgggggag 720 gagaaaaggg gagtccagtc gtcatgactg agctgaaggc aaagggtccc cgggctcccc 780 acgtggcggg cggcccgccc tcccccgagg tcggatcccc actgctgtgt cgcccagccg 840 caggtccgtt cccggggagc cagacctcgg acaccttgcc tgaagtttcg gccataccta 900 tctccctgga cgggctactc ttccctcggc cctgccaggg acaggacccc tccgacgaaa 960 agacgcagga ccagcagtcg ctgtcggacg tggagggcgc atattccaga gctgaagcta 1020 caaggggtgc tggaggcagc agttctagtc ccccagaaaa ggacagcgga ctgctggaca 1080 gtgtcttgga cactctgttg gcgccctcag gtcccgggca gagccaaccc agccctcccg 1140 cctgcgaggt caccagctct tggtgcctgt ttggccccga acttcccgaa gatccaccgg 1200 ctgcccccgc cacccagcgg gtgttgtccc cgctcatgag ccggtccggg tgcaaggttg 1260 gagacagctc cgggacggca gctgcccata aagtgctgcc ccggggcctg tcaccagccc 1320 ggcagctgct gctcccggcc tctgagagcc ctcactggtc cggggcccca gtgaagccgt 1380 ctccgcaggc cgctgcggtg gaggttgagg aggaggatgg ctctgagtcc gaggagtctg 1440 cgggtccgct tctgaagggc aaacctcggg ctctgggtgg cgcggcggct ggaggaggag 1500 ccgcggctgt cccgccgggg gcggcagcag gaggcgtcgc cctggtcccc aaggaagatt 1560 cccgcttctc agcgcccagg gtcgccctgg tggagcagga cgcgccgatg gcgcccgggc 1620 gctccccgct ggccaccacg gtgatggatt tcatccacgt gcctatcctg cctctcaatc 1680 acgccttatt ggcagcccgc actcggcagc tgctggaaga cgaaagttac gacggcgggg 1740 ccggggctgc cagcgccttt gccccgccgc ggagttcacc ctgtgcctcg tccaccccgg 1800 tcgctgtagg cgacttcccc gactgcgcgt acccgcccga cgccgagccc aaggacgacg 1860 cgtaccctct ctatagcgac ttccagccgc ccgctctaaa gataaaggag gaggaggaag 1920 gcgcggaggc ctccgcgcgc tccccgcgtt cctaccttgt ggccggtgcc aaccccgcag 1980 ccttcccgga tttcccgttg gggccaccgc ccccgctgcc gccgcgagcg accccatcca 2040 gacccgggga agcggcggtg acggccgcac ccgccagtgc ctcagtctcg tctgcgtcct 2100 cctcggggtc gaccctggag tgcatcctgt acaaagcgga gggcgcgccg ccccagcagg 2160 gcccgttcgc gccgccgccc tgcaaggcgc cgggcgcgag cggctgcctg ctcccgcggg 2220 acggcctgcc ctccacctcc gcctctgccg ccgccgccgg ggcggccccc gcgctctacc 2280 ctgcactcgg cctcaacggg ctcccgcagc tcggctacca ggccgccgtg ctcaaggagg 2340 gcctgccgca ggtctacccg ccctatctca actacctgag gtgagggccc gggacggggc 2400 acgcccagcg cgtccgggag tagcggttcc gttggcggcg gcggccgcca accctcagcc 2460 ccagccccag cgcaccgctg cgctccccgg ggcggccgga gagggtgggc agcgggacac 2520 agcacagggg cagttgcctc ccttcttctt ccctcctctc ctcactcttg gggacacgaa 2580 ggtgggcgca gaatatacta tttttggggc gtgcctccct gaaagctgtt tttttgtttg 2640 ttttttaact ttccgaatct tccagattcc gaagcagaac caaccccgat ttaaaacgtg 2700 cagcgtcaca ctaggtccgc tgtagcccag tggggcagaa agtgcgcggc gagttggggg 2760 ctttatgaaa tgcttctttc ttagaagaag gacgtttacc aggagtgctt gtcttggaga 2820 ggagttaagg caccgttccc ccgggagggg tgggacttga gaggtggccg gccagaaccg 2880 aaagcagcac catcttaggg atttgaacac ttcagtggct cagttttctt aagaatctca 2940 agattaaaat taagttcacg tgggaaatgt ttaaactgtg gatttaaacg cctgtcactg 3000 cattgcaccg ttttcttatt attgcttgct attcactaca atttttttta tatacaggtt 3060 taaaaaacac tactttgcat actgaagtaa tggaatgtaa aaaaagaatg ctctgtttgg 3120 aatcttatgt tgtgaatagg caaaacagtg tcagtgtatt ggacaatact ttaaaatgac 3180 aaacatatac ttgcttaagt aagcaatgat tacagggttg tgttttaaaa actcaaaacc 3240 aaaacattgc aaagtaccat cgaactttta aagccaaacc atatttgttt tgacccagca 3300 tacagacagg aaggacataa catttcattt gtcaaagact aaattgtttc tatataaaga 3360 gttttgtaga aagatttcct tttaaccgac tttaactttc taggacataa tattatacac 3420 taattattgt tcttttatat tggtgctact gatgaatggc taatcatttg caagtatggt 3480 gaatccagtt acggatagtc tattaccaag tttagtttgc atgtctttca agtgtatata 3540 tacagttctg tttttaaaat ctcctttcac cctgttaata ctggtttaag aaacctttag 3600 tattagatag tggtgcactt aaaaataaat ggagtacttt gttttgcatt tcaaggccgg 3660 attcagaagc cagccagagc ccacaataca gcttcgagtc attacctcag aagatttgtt 3720 taatctgtgg ggatgaagca tcaggctgtc attatggtgt ccttacctgt gggagctgta 3780 aggtcttctt taagagggca atggaaggta ggctcctttc ccctgatcct ttattattgg 3840 tttaattgta aatggagacc atctaatatt gtatagattt caattattcc ttgtttctta 3900 taagaaatgg tgatatttcc atataattta aaatatatga tgacatttta acaatatgtt 3960 tttatttatg atactcaaaa tggaatgtgg ttgggtacta taattgcata ctctttgact 4020 aacactttca gtattagaca taagtcataa aaatcttgag gacagtgcta ctattgttcc 4080 ttaactgctt agctttgagg aaacagcttt gttctaatag tacttttata tatctattat 4140 gtaggtatat gtgtatgcag tactttaaaa ttttgattaa aagaaaaatg gtagttgaca 4200 catatgtaca tgtatgcgta tacatatttg tacatacacg aacatatatc acaacatgta 4260 tatgatgcag ttttctacat gatactgtct tttgactaca tgaatattta tgtaatattt 4320 acaaagaagt aattctaaac aaatttttga attctctttt tgttcagtat atttttgtaa 4380 gtgtataaga ggacaggatt aaacagttta aaataaaaaa cctggacatc acagtaacat 4440 aatttctaaa gaagtatttt gctttaagta aaactttcat gtttttaaac tcattgaact 4500 tacatgctta atgatttcag atttacttgc atagtgtttc agattttaac tttcaaagaa 4560 aatatttttg aatttctttc tacctaaagt ctaagcagcc aaacatcttt acatttgaag 4620 ataaaaatac attgaaagat ttcatatttt aataccagca ataaatgtat tctataacta 4680 tgtaaaatga agcttaggat tccctctgga gtgctgagat cacacctaga caaggaacca 4740 aggatctgaa tgttggcttt ttgtttcctg ttctgaggat ttttgtttgt ttgtttgttt 4800 caacagccct ctccttacaa gcagaaaagc aggtagtagg aaatttcact ttaagggagc 4860 tttcaaagga gttcttcata acaaatattt gctttgtata tttttagaac atgatttttt 4920 ctcacaaaag atgaatgtat tttactgatg ttgaacatat tcagctttag ggggtttgat 4980 tgcattttaa actaattgag gcagtgttaa aagtggtact tgagaaaata gggcaactga 5040 tagtggctct tacccattga cattatttat ttacagttac agttggaagt tctttgtgtg 5100 gaaaagtcag ttttccaatg ggtaattgga gttaccattt ttatctgact ttggttctgg 5160 tttcttaaac attgccttct gcattaatat gatttcctct cttcttaaag tctcctaagg 5220 atagtataat tttataagtg aatgactccc ttagaatcct cttaagccca atttgtccta 5280 ccccagctcc ttcttttgaa agataagcaa atctcaaaga cataaatatg agtttccaaa 5340 ggtcacaaaa ctagttagta gctgattgtt agtaaacata ggttaaatat ttttacattg 5400 cagcgcttgt aaatcagaga tgatatgcaa aagtagatat ataaactgtt tgattcacag 5460 aagttatttc ataaagtgca tatatagaac aaagagcacc ctaactaaaa tacaaatgct 5520 ttctcgtcat tttgttagaa tagcatccaa aactgtagac gaagtctttc caaatgtact 5580 cttagaaagc atttgttgga ctccggctgt tggcatggtc ctatagtctt gagtactaga 5640 agtgaagcac ctttatttag cagtaattac aaagagttac ttaagattga tgcagataaa 5700 tcattcatga aactagaaca agattatgaa ctacattagt aagttccttc attcagcaat 5760 ttatgccaaa gatacacttt ccctgacttc acttttctct gccttgagat aaaatgagga 5820 taacagtggc tatttcttag ggttgctata aagattaaat gagctgatac ttgtaaagta 5880 tgtaaaagaa ggcctgacat attatcagtt tccattgaca tttctacttt caaggaactt 5940 gtaatatagt tagggaggta acatatgcac ataaaacatc taaataaaga ttctcagtaa 6000 atgcccaagt aagcaattct gtaatgtata tgagatctgt gtggtttgtg agtttttgta 6060 tttggacaga gcgaggtggt tatgggttga aatatgtata ttcttgaatg atgaagaagt 6120 ctacatggaa gatatgaaca tttgattagt aaaggacaaa taagctttct aggcattgag 6180 gagagcttta agtgtataca gtcaaagaag agtgaagaaa ttaagattac actgactaag 6240 cattgaatgt tcacattagg aaattgagag agttaaagtt tgagaaacta gattgctagt 6300 gtttgggtga atttggagaa tcggtaattt aaggcaagag aatatagaga atgttctagg 6360 agttttcagg aatgagaaag ataagtagaa ggacttatat caggttcaaa atcttcaata 6420 aagcaatgct gcgtgatgag ctggttcaag gtggcgctgt gtgtggatgc caatatggcc 6480 agaagtttaa agtaaacagg caacaatatg gatactaatg ttgccaagga tgagaatgaa 6540 aatgatggca ttataaaatg ctttctctgt gccagtgact atttcagtgc tttgcaggtc 6600 ttaacttatt tagttgttat aatgttggta ttattattat tactcccact ttacaaatga 6660 ggaaactaag acctgtagat gttaagctgt cttaatacgc ttaggaaatg gtaaagtcag 6720 gattcaaatc caggtggtat gaatccagaa tcccggccct tgaccactgt gcttcctttc 6780 tcataatagg aaatgcagtc aaagaaaaac aatagagggt tagaagaaaa gatgtgagcc 6840 aagtgatgaa acatctagga aggtaaaagt gaatcctaaa ggagaatgca agagcagggg 6900 taatgagaag tgtgtgatat aaaaggatgg atcatattaa cttcacattt agggcagcaa 6960 taagaagaca taagcaggag ccaacagcca gtttcttcac ctccctccca tgttaggaga 7020 gggaggtgat aaagcctata aagtcattct ggatgactgg gtttccctgg atatgagaca 7080 gaagagagaa gagataaaga aataagatac cactcaggaa atgggagaaa ggagttggga 7140 aaaatgattt cttttaagct aattgaactg tttaggatat agtattggcc aaaaaccagt 7200 tggttggata gcccattgca ttcctttacc aagacttgta ggtttggagt gaaccatgaa 7260 aggaccagga attgacttaa tgccttccaa agagaggaag taatcatgac ctgccagtcc 7320 tacaaatgca gactactaac ctggtatgat gaaggaaagg actatttctc aatggcttat 7380 ctttgccagt acacagtaac cagcccagtg ctaggaatat actaggcatc cagtagataa 7440 ctgctacatg atccagtcat cataactgat aacgccacac ttttattttt tggatgcttt 7500 actcagtgac agcatttgtt gtgaatacat ttggtgtaat atcattaaac acatgttata 7560 atacaattga aatgtattac ttagaagaca ctaagctaag taggtattga aggattttca 7620 attgtattgc atattatgct cacttttttt tttttttttt tgagacggag tctcgctgtt 7680 gcccatgctg gagtgcagtg gtgtgatctc ggctcactgc aagctccgcc tcccgggttc 7740 atgccattct cctgccttag cctcccgagt agctgggact acgggtgccc cccaccacgc 7800 ctggctaatt ttttgtattt tttagtagag atggggtttc accatggtct caatctccta 7860 acctcatgat ccgcctgcct cggcctccca aagtgctggg attacaggcg tgagccacca 7920 cacctggcct gtcctcattc ttttattcat atattaattg ttcatgaagt aattacctta 7980 attacattag ttcatgtatt tattgagtac ctgccatgcg ccaggcacta tgttaggtac 8040 tggtgaaacc acagtaaaac gagagacctt gctggctgtt gactaaagtt tatagtgtgg 8100 tggtggagag agacatttta cctatttgtg tacacatgac taattgcaca tgtggtaagt 8160 gattcgttta tgcaatgttt ggacaactag agaattgacc cctctgtcag ataatgggga 8220 aagtttttca gacctagaca tcacaataaa tagactgcag aggaaactag acagaaatga 8280 aatactttta tgaataaagt gtttctttca aaagttgggt atacttggtg tcataggcta 8340 gtaatgaaaa ctggtttggt agcatagttc tccttgatac agcatcagaa agagaaagat 8400 tgaacatcca atttttcatc agcaacttct gtgatttatt tgttctatat ttagagctct 8460 gtaatgcttt ctccatcttt tgtagtgcac agatcatcca gcaatctctt ctactataat 8520 ctatttgaat ttgaataata tttgggctct gagaagatat tttgccaata atcttattta 8580 tattcattta atctccaata gagtctgctc tatagcagct ttcacattac cctaaaaata 8640 aatatgtagt aattcactag attttatttc aaaactctat atccaatttc ttttcaagct 8700 gaggtctctc agtttactat ttagcatgga taatgataga ctggttttaa gcaccacttt 8760 acattagggt tcattgaaat ctctactgta ctgaaaaaga aatggttaaa aagatagcat 8820 ttggtgtcat ctgttcatat ttggttattt agagctccca gatttttaat actctttcag 8880 aacatgtact ttaataccac tacaagagcc agaggagaag cagtggtatt agccatgcgt 8940 gctagtgcta ataactgctc ctgttctggc cactgaatca ttgtacttta attactcaag 9000 taacacaaaa atccatctcc tttaaaaaat gaaaatgtta tcaataaggc ttaagtcctc 9060 ttgatcaata cctctaatct gttcccttgc ctaccatctt tctatacctc tacctcagtt 9120 tttcccatgt gtatgtgtgt gtggaatatg ttctctgggg tatgttaata tataaatggt 9180 attatatatc ataatttatg catcatgtta ttttgataca aaaatgactt gaagctctat 9240 tgttcatata tgtagttcca acttgttttt ttaaccactg cttaatattt cataatatga 9300 aaaatcatta tatttaggaa ttactaatta cactattact tgagtaaacc ctttttgtac 9360 atttctcctt tggtacatgt cttgcttctc tttccagaat atattgaagt attttgatac 9420 agttctaaat gaactagatg attagctttc tacctgctgc taagatgatt tgattcacta 9480 atttattcac ttcagataca tgtattgatc ctacatttaa aagttgcttg tgctgggact 9540 gaagatgaaa atgtatgcag accctgtctt tgacatgcac aaaaccaaga agaagaaaac 9600 aaaacagaac aaacctgtgg tacaactagt gaacatgaga ataccttgat acaagttatg 9660 ccagagccgt ggccaacaca ccgtagtatt cagttagaca catggagaag aatggcagtt 9720 tattctgcta acatatcttg atgttgtgca agaggcttat tcatttaata gtcacttaag 9780 tcgccttcac tataacccag gcactgtttt aactgcttca ctggattagc tcaattaatc 9840 cttgtaagag tcctttgagg tggaggttct gttagtatct atattttaca gatgggtgta 9900 aagagaggtt tctactattc aggaggtctg actctaactt ctgtatctta aattcctgct 9960 tctgtgctgt tttcctagac tgttttgtat ttgagcaaat attaattact ttatactttt 10020 tcagataaat acatggtctc ttataggaga ctggttttca aattgtgctc cttaactcag 10080 tcaagttcct tcttctattc agaacagctc tgtatttctt tatttggttt atatttccac 10140 ttgagatttt tattgggaca aaggattctc agccaacctt tttttaaaaa gcactccttt 10200 ataacaggaa acccttagat ctcaatcaag tatttattcg agtgactcct aagaagtttt 10260 ttggctctat ttgctggtat ctttttggca tttgtgcaaa gacgagatga ccatttgtga 10320 tcaagattta aagtccaact gctcactctt ctaacagcat ggccacatta ttagccgaaa 10380 atcacatctg agttatagag cttttgcttt tgtcagaaaa aaaacaaagt aactccaggg 10440 aatcttttat cagtcacttc tttaaaggat tgattgaaat aattgacact taagacagac 10500 actcaaaaat aggcacacta atcatttaca caagggattt ccaaccccta ggatgtggac 10560 tggtaccaca gccagtccat ggtccggtgc gggcccatgg cctgttagga agtgggccac 10620 aaagcaggag gtgagcagtg gccaagcgag aattacagac tgagctccgc ctccctgtca 10680 catcagcagc agccttagat tctcataaga gtgagaaccc tattgtgaat tctgcatgca 10740 agggatctag gttgcgcact tcttatgaga atctaatacc tgatgatctg tggtagaaca 10800 atttcatcca gaaaccatcc cacaccatcc atggaaaaat tgtattccac aaaactggtc 10860 cctgatacca aaaaggttga ggactgctgg tttacatctt ggtttttaaa ctccattgtc 10920 atttaagaac atcagaaagt cacatactct gtatatcttc cctatctacc taatttgagg 10980 agaggccgat gaacacaata tccttcttgt ggctagatgg ccctcatata attaactata 11040 taatgcatac ttcatataat ctatcatcaa gacatttttt tctcctctga tgagatatta 11100 tgctttttaa taagtgatat gtaaatagcc tattgggttt tcctttttat tttaactcct 11160 gcaaaacaag tacattttaa tgctcaattt taaaaaatta acaggttttg agctttattt 11220 ttaactgcta ctacagcgtc ttgtgttgtg tactttatat gacattttaa ggaatcataa 11280 cttttttctt aaagcatagt atttactagt aatcattatt tctttaggaa aaaaatgcaa 11340 atacttttca tgctatttac tcttctataa ccaagtgatt tatttatgag atagaataca 11400 catgtagtga gtatggaccg tgagctcaaa acagtgctgt agcatttgta cgaaatataa 11460 aaattaaagg gtaaaaataa tggttatcat ttattgagtg tttactaaat tcaggtactc 11520 ttctaatgtc tctttgttta ctaactcatt taactcttgc aacaaaccta tgagacaggt 11580 tctatttttc tcattttaca gataagattt gttcagttag tcaacagtag agcagaactc 11640 cactcctgga gtcaatgccc ttaacttcca ctggatattc ctttgcagga aaagatacag 11700 tctttgattt caaggagcct gcagtgtgtg tgtggaagtg agtgattaac agacaacact 11760 gcaaatttag tgtttgatta aattgtgtgt tgcagactta gggtgctata agtgaactag 11820 agaggaaagt accaagagtt gtcattactg attgcctata tataatagaa accatgctaa 11880 atgctttata tttaatattt ctattcttca caacaatcct aaaaagtaga atttaccatt 11940 tccacttcat atgtaagcaa agactcagaa gttagttaac ttaccccaat taatagtagt 12000 actggcagag atgtgtctgc agtagttaga aaaggtagtt tattgagcta gtaagattta 12060 ttcaaagcct ttaaaaacag agacagtaat taagaaacgt ttatttttga tagttttttt 12120 aatataataa tattgtgact cctggcgtat tatacacaag aaaaatttca ggtgggtcaa 12180 caacttaatt ttttttttat ttgaagaaaa gaaacctaaa agtatggaaa gatgatggag 12240 gtatgtattt atagtcttgg gatggaaaac tttctaaatt tgatataaaa tccagaattc 12300 aaaaacaaat ttgctccatt tgctatgtaa tctcaaattt cttatataag gcaaaaaatt 12360 cataaagtca aagacaacaa caaattagga aactgtaaca tatatgtaaa tcaaaaggaa 12420 attattaccc atatacaaaa tattcttaca aatcaagaaa aagacaaaca ggaagaaaaa 12480 tgaacaaaaa atattaagac agttcacaaa gtacagccca cagaagtatg aagaaggctc 12540 aactcactaa taagcattac tgattaaaat aataatggga atttcagcaa tttgagtact 12600 ggaataataa tgaataacaa ataatgacca ttggagaatc tgaagatagt ttggtattgt 12660 caattaaaat atgcgttact ttggcccaac aattctactt ctagaaactc atccttacag 12720 aactctagaa tacctgtagg tggatgttta ttgcagtatt acattattta taatactgag 12780 aaactgaata cagcccaaat gtctgttaaa aagagattag caaattgtgg tatacccttg 12840 caatgaaaag agtgagttag ctgtctatac tacagatgtc aagaagtctc taaaatattt 12900 gattttatga aaaagcaagg aacaatatca ttctgggtaa aaaattgtac ccatttgatc 12960 ttgctaggta tagagagcta tctaaaagaa aataactaag tgcttactgt caggatgaga 13020 gacttccaat ttctgatgac atgttttggt cttttttttt tttttttttt ttttgaggtg 13080 gaatctagct ctgtcaccca gcctggagtg cagtggtttg acctcagctc actgcaacct 13140 gtgcctccct ggttcaagtg attctcctgc ctcagcctcc cgaatagctg ggattacagg 13200 tgcccaccac catgcccagc taattttttt atttttagta gagacagggt ttcaccgtgt 13260 tgaccagatt ggtctcgaac tcctgacctc aggtgatcct cccgccttgg cctcccaaag 13320 tgttgggatt acaggcatga gccactgcac ccagcttcat tctttaagca agtatttact 13380 gaaggggctg caattacaca ccaacttggt gcctgacttt cctccacact ggccccgcct 13440 agtctcagct agcctctgct gccctaatgg tgtccagtga ccatatgtgc cacttttcac 13500 cttcaggtac cctagaatgt tgaaagagac taacaaagaa tgaaacttga agaatattga 13560 agcaaaagct tgaggttact ctgtatcatg aattaaggaa tccttcctga ctagatggag 13620 ccaagaatgg acacaggccc acaattcctc acacacaatc tcaatattca aaaagcactg 13680 caaacaaaaa ttttgttgta gtcacttgtc aacaatacct aacctgacgt gaatttgttt 13740 ggcagcaaaa tctgatctga aatcacatga ggaaatcttg tagtctatgt aaatattcgt 13800 acattttgct gcataaatat aatgttttat taagtgttgc tctattttat ctttctaaaa 13860 tacaaacaat tttgaatttt gaaacacaat tggcccaata catttcagct aagagatttt 13920 ggacatgtac tcagaaatga tttatagact atgttgagag tggtgcctgc tggctttttg 13980 caatgtagca gccctgaaaa tttttataaa caattcttat catttattct tatctttttt 14040 ttttgagagg gagtccccgc tctgtagccc aggctgaagt gcagtggcac aatctcagct 14100 cactgcactc tgcctcctgg gttcaagcga ttctcctgcc tcagcctccc gagtagctgg 14160 gattacaggc atgtaccacc acacacacta atttttgtag ttttagtaga gatggggttt 14220 caccatgttg gccaggttgc tcttgaactc ctgacctgaa gtgatcctcc caccttggca 14280 tcccaaagtg ctgggattac aggcgtgagc aacagcgccc agcctattct tatcattttt 14340 aataaattct tagtaccatg ctgggttcta cagggagcta taaaagataa ataagccata 14400 ttacctaact tcatatagtt ttcgttcaat catggagttt atcccatatt caagtataat 14460 ataggtaata tataaatcaa atgtgttttt gatttcaagg ggaaaaagat catatttagc 14520 tagggagacc aaaatgtgtt tttatggaaa ggatagcatg ctagatgggc tgctgagaat 14580 gcatgaaatt tttgatagca agaattgtaa tggcttttgt aaacaaaaac tacagactga 14640 gcagaaggac aatttggcta aagcatggaa catggtcaga atcccttacg attatatata 14700 aattatttgt gttttttcta ggataattct tttcatgagg ttttgaaaga gatcagtgac 14760 cccttattcc cacccccaaa aaagttagct taccactgct ttaaaggaat ggtgaggatt 14820 attataaaac agaaggtcaa gaaaatgtgg aatgcacaat ggggtgttta caattaattg 14880 agtaaagagt gagaagcccc taaatatttt ttgggcagtg tagtggcata ttttaatatt 14940 taagaaaatt agtatatcag cagtatattg gaaagattaa tgtcagaaag gtaagggaca 15000 aggaaaacag tctggggatg gtgcactaag gacctgagcc agggttgtgg agagaggaat 15060 gggttcaatg ggtagattgg aaccaattga gaagggtgat gaaagcgaca cagagaattg 15120 tgggggtggg ggacgtcacc ttcagaaata ggaagtacat gatcatttta ctcagtaaag 15180 gagtgatgtt gaaagtagta tttaggaagg attatcctaa aagcaccatg cagaatatat 15240 ttgtacagag attagatagc ttgaagtcag agagaacagc taggaaactg gtaacacaga 15300 ttctgagaag tagtttaatt tcaaattgtt tgtattatat tctacataat gtcctcagca 15360 ggatattgaa ataatataat ttttgtgcac aaaataactt atgagaattg agatgttctg 15420 gtttgtcact tcatttagtc gtctgcttat ttattaattt ataccaaaaa taatgccgtt 15480 ttgtgcctgg atttgtgtta aatattgaga ctcaaaaatg gatgagccaa gttcataact 15540 taggagaaag gcacataaac acgttgcaga aatgcagtct ggtaatatct atgagaggtg 15600 tgagctgaga attgtggaag tataggacag gaagatctaa ccctggtgtt taaatgcggg 15660 gtatggaagg agaaagtctg aatcctgact aagtattaga ctaaaaatat taaatcttaa 15720 aaattattgt aatttcaaag tttgtggcaa cttttttgta attaacagtg gagaccacaa 15780 tggtttatct tctttaagca gagcacttca gtgagtgttt ctttgtgcaa aggcccatcc 15840 agaggccact ttggatgggg gggcggatga aagtatgaag aactgaacct ctctatgtac 15900 tattttaata gaaattaggg aattgtaggc tagcagctgg ggcaaggagt aggaaataga 15960 aagggcctaa caactgagta ggaagcaagg gctcaaggag agtagatgcc tgtgagaatt 16020 tccctattgc aggcaacaat ccataaatcc ctaggctggt agtgcctgat agcaaacttg 16080 gaattgttta ctatctcatg ttttaattac ctccagctac ctcccaaccc ttaagtaaat 16140 ttttgctgtt tacattttcc tttataaaag gatatgcatt tcagattgct gacctttcct 16200 atgatgacaa gtgtctcagt agtatgatac ttagaagata tataggccgg gtgcaatggc 16260 tcacgcctgt aatcccagca ctctggaagg ccgaggtggg cagatcacga ggtcaggaga 16320 tcgtgaccat cctggctaac acggtgaaag aaaccccgtc tctactaaaa atacaaaaaa 16380 aaaaaatagc caggcatggt ggtgggtgcc tgtagtccca gctactcagg aggctgaggc 16440 aggagaatgg catgaaccta ggaggtggag cttacagtga gtggagatca caccactgca 16500 ctccagcctg ggtgacagag caagactctg ggaaaaaaaa aaagaagata tttagcagaa 16560 ggtacagtta atctgcccct tcaagttaaa gataatgttc atgatagaaa aaaggaggta 16620 acttctgttt gaaagacact gttgttttgt agatagcatt ttgaaaacaa gtctttgggg 16680 atgattccac tgttatttga ttttgttgtg aaggtattat aagagatcaa aattttatag 16740 tttcatacat aaacattttg aaataagatt ttctaacttg ctaaaaaatc ttccaaaaaa 16800 attcagttag gttctgaaca catttcataa tagtggcatt tattgaacac atatgtcagg 16860 aacttggcta aatgtgttat attaaacaca aagattttaa atcacttgca cagataaata 16920 gtaaataaaa tggtagagct gagattggaa cccacagaac cttctctctt atttgttaat 16980 attacctctt attcattatc tagtgtctct ttgtcctaag tatttgtaat gaaaattgat 17040 tgacatcagg gagagtagat acttgctagc tgaatttctg gggtaaaaga agcttttcat 17100 aactggtatg tggaattgaa agtgaatatt atgactagat aatctgccct gcagacagtc 17160 ccctcattct ccatggcttc tgggatggct gtcttccacc ttctcttctc agcctgccag 17220 cactcccctg tcctcacttt cagctaacag ccttgcttcc taattttcgg aaaaaaactt 17280 tctgaaattg ctaccaccat ctgtatggac agaagtggcc tctgaactat aatcccattt 17340 ttctggggtt accagagctc tgcagttctc aagtccagcc ctctctcctt gaccactaga 17400 tccagattac tcctgctgac tcaaggacag gctctaccaa tcctcccgca acatttaaat 17460 catttttccc cttctctact ggataattcc tgtcagccta caacatgctc ttccttttct 17520 catcttaaaa tataaacaaa ctacttttgt tgatttcact tactcttcag ccaccaatca 17580 tttttcttct tttacagcaa aactcttcta acacattttc ttctcccatt cactcttaaa 17640 cctaacccaa tcagttgttg acctctgtca ctccaccaaa acttctcttg tcaaggttac 17700 catgttacta aatcttatgg gcaattgtca gtcccatgtt acttgactca ttggtagcat 17760 ttgacaattg atcactccac cttcctgcaa atactttatt cgcttggttt tcaagacacg 17820 accctctctt gattttcctc actagctccc cttcaaactg ttcttctcct ttagctttta 17880 atatgagagg gtccagtgct gagtattctg agtatttggt cctcttccct tctgtttata 17940 tacttacttc cttggttatc taatccagtc tccaaactgt ggataaacca cccaaaatcc 18000 aatcacttcc aaatttctat ctccagccca gacctttccc ctgaactcca gactgaatat 18060 ctcaaaccaa cacatcccaa agttggctcc tgatctactt agggttttct ctggtaagcc 18120 agtgttcttt tggtcctcaa gctatctgcc tcagaatcac ctgggtactg gtttgagatg 18180 atttctgcac ctcgccccag gtaagccaag tcaaaatatc taagaaaggg acccagaaat 18240 ggcatttcag ctagctgttt ggttgtttct taaagttctg cttggctcag aggggagcca 18300 gatacaaagc tgagaggtga aatggttggc tttgtttttg agaacattaa ctctagaagc 18360 aatggggaat agaggcagaa ggtcatgcca gggagatcag acctaagatt atttaaaata 18420 ctctttgatc gagatctggg ggccttcagt cttatgctga gaaaacacta cttttcattc 18480 cctaaagcag tgaacctgaa atatagaagg caggcaggct gtggccccta ccaagaatgc 18540 tcctttctct aggaagaatt ctaaaaaatg tgagaggcta gggagggagg ttggagactt 18600 ccactttttg tgtccttact atataccagg cactatagta gacacattat atgtatttgt 18660 tataggcata ccttagagat atcgcaggtt ctgttctaga caactacaat aaagcaaata 18720 ttgcaataaa gcaagtcaca caaatttttt gctttctggt atctaaaaaa gttatgttta 18780 cactatactg cagtctgtta agtgtgcagt agctttatgt ctaaataaac aatgtacata 18840 ccctgattga aaatacttta ttgctaaaat tgctaatgat cacatgagcc ttaagggagt 18900 tgtaatgttt ttgctggtag agggtcttgc cttgatgtga atggtcattg acttatcgag 18960 gtagtgattg ctggaggttg gggtgtctgt ggcaatttct ttaaataaga caacagtgaa 19020 gtttaccata tccatagact gttcctttca caaaagatta ctctgtagta tgcaatgctg 19080 tgttttaccc acagtagagc ttctttgaaa attggagtca cttttctcaa aacctgctgc 19140 tgcactatca cctaagttta tataatatcc taaatcctaa atcttttgtt gtcatttcaa 19200 caatgctcac agcgtcttca ccaggagcag attgaatctc aagaaaccgc tttctttgct 19260 catccatgag acaagacccc tcatgtgttc aaattttatg agattttagc aattcagtca 19320 catctttgaa ctccacttct aattccagtt cttttgctat tcctaccaca tatgcagtta 19380 cttcctccat gaagtcttga atccctcaaa gtcatctata agggtttgaa tcaacttctt 19440 ccaaactgct gttaatgctg atgctttgac tttctccgat taaccacaaa tgttcttaat 19500 gacatctaaa atgatgaacg ctttctaaaa gattttcaat gtactttgcc cagattcatc 19560 agaggaatta caatctatgg cagctgtagc ctaataaaat gagtttctta agtcataagg 19620 cttgaaagtc agaattactc agtgatccat gggctgcaga atagatgttg tgttagcagg 19680 catgaataca ttaaccttct tgtacatttc catcagagct catgggaatt tttttttcta 19740 agcagtaggt ctcaacagtg ggcttaaagt agttagtgga ccatgctgta aatagatgtg 19800 ctgtcactca ggctttgttg ttccatttct agaacacaag cagagtagat ttagcataat 19860 tcttaagggc cttaggaact tcagaatgat aaatgaggat tggctttaac ttcaaatgcc 19920 cagcttcatt agcccctaac aagagagtca gcctttcctt tgaagctttg aagccaggca 19980 ttgacttctc ctatctagct ctgaaagttc taaatggcat ctttttccaa gagaagactg 20040 ttttatctac attgaaaatc tgttgtttag tgtagctacc ttcatccatg ggcttagcta 20100 gatcttttgg ataacttgct gcagcttcta catcagcact tgctgcttca ccttgtactt 20160 ttgtgttatg ggaatggctt ctttccttta acctcataat ccagcttctg ttttcaaact 20220 tttcttttac agctttcatc cctctctcac ccttgataga attgaagaga attatggcct 20280 tgctctggat taggtttggc tgaagggaat gttgtggctg gcgtgatcct ctatccagac 20340 cattaagcgt ttctccatat cagctataag gctttttttg ttttgttttt ttgctttctt 20400 atcattcatg tgttcactgg agtagcactt tttaacttcc ttcaagaaca tttcctttgc 20460 atttacaact tggctaactg gtgcaagagg cctagctttt ggcctgtctt ggcttttgac 20520 attcctccct cacgaagctt aatctttcta gcttttaatt taaggtgaca catatgcaac 20580 ccttcctttc acttgaacat ttagaggcca ttgtagggtt gttaattggc ctgatttcaa 20640 tattgttgtg acacagggaa taggaaatca tgaagaaaga gacaaggaca gaatgccaat 20700 tggtggagca gtctgaacac acacatttat cagttaagtt tattgtctta catgggcaag 20760 gtttgtggca cccaaaaaca attacagtaa taacatcaaa gatctctgat catagatcac 20820 catagcagag atgataatga aaaagtttga aatattttga gaattactaa aacgtgacag 20880 agacaggagg tgaacatgtg ctgttggaaa aatgatgccc atagtcttgc tggatgcagg 20940 gctgccacaa accctgaatt tgtaaaacat acaatatttg tgaagtacaa ttaagcaaag 21000 cgcaataaga tacgatatgc ttgtatctcc cttaattttc agctaagagc tgtttgagaa 21060 gttactgcca tctccttttt tcagatataa gaattaaaac aaggaaggaa gttaactttc 21120 ctaaagatac ctagccagtt agtaatggag ctggtaacca aacccagttc tggcagactc 21180 caaaaattaa accgctttca ctaaagcaca ccactgggca ctgtgtttga agcacgagat 21240 gatattagca actgattgaa gatttattga aaagacttaa aactcttatt gtagtcaagt 21300 cgtttaaaaa tattttttac ttttaaaatt tattattatt tagagacagg ctggagtgca 21360 gtggcatgat catagctcac tgcagccttg aactcctggg ctgaagtgat actcccacct 21420 caacctccag atattttttt aatagacaag gctttgcttt gttgcaggct ggtctggaac 21480 tcctggcctc aagcaatcct cccaaagtgc tggagtttca ggcataaacc accttgccta 21540 ggctcatttt tttttttttt tttttagacg gagtctcatt ctgtctccca ggagtgtaat 21600 ggcacaatct ctgctcactg caacctctgc ctcccaggtt caagtgactc tcctgcctca 21660 gcctccagag tagctgggat tacaggtgcc caccaccaag cccacctaat ttttttattt 21720 ttagtagaga cagggtttca ctgtgttggc taggctggtc tcaaactcct gaccttaagt 21780 gatctgcctg cctcagcctc ccaaagtgct gggattacag gcgtgagcca ctgcacctgg 21840 ccagttcaaa tcttatatat tttggtgcct agacttaaat aacatttagg aaaatgttaa 21900 ttaattatcc atatagttct acctttttct ttcttttttt ttttttttac aaagccagtt 21960 tcaagtaaga ctggggagat tctagaaagt tataacagaa tcttaagtca cttgatatgc 22020 ctaaacagaa tttgaagaaa taatgaagtt gaacacattg ataaacttag gatttctgta 22080 atcaggaaga aaaaactaaa cttcatagat atgagggaag gaattaacca tttgcatgta 22140 tgtaactgtt ataatcctac atcagagtag cacaaggaca tgaacaactt tgttgacaaa 22200 tagttttgta attttttaat gttttttatg tagctgtctt aaactccttt ctcaatagta 22260 tgtgaggtca aatgattgtg gtttcttttt aaaattttgc atgtaatgtg tagttcctta 22320 cattctctaa atgcttactg tttagctaac tatgttatta tggatgtcaa ttataactaa 22380 gtacttcata caatttcacg ttttataagt tagaagatat gtgaataatt agggcatttg 22440 gaagagttcc agagagattt ttttatttag aaagagagga gcagaacagc ttcaggaaag 22500 atagatgaaa agctgattca ttcaatttaa gatagttgaa gggaagaaaa aagaacacat 22560 tatttagaat gatgacatga agaacttaat ttcctttttt tacttatact ttaagttctg 22620 gggtacatgt gcagaacgtg cagttttgtt acatagatat acacgtgcca tggtggtttg 22680 ctgcacccgt caacccgtca cctacattag gtatttctcc taatgctatc tctcccctag 22740 ccccccatcc cctgacaggc ccaggtatgt aatgttcccc tccctaagtc catgtgttct 22800 cattgctcaa ctcccactta tcagtgagac aatgcggtgt ttggttttct gttcttgtgt 22860 tagcttgctg atgatggttt ccagcttcat ccatgtccct gcgaagaaca tgaactcatt 22920 cttttttatg gctgcatagt attccatggt gtatacgtgc cacatttgct taatccagtc 22980 tgtcattgat ggacatttgg cttggttcca agtctttgct attgtgaata gtgcttcagt 23040 aaacatatgt gtgcctgtgt ctttatagta gaatgattta taatcctctg ggtttatacc 23100 cagtaatggg attgctgggt caaatggtaa ttctagttct agatccttga ggaattgctg 23160 cactgtcttc cacaatggtt gaactaattt acactcccac caacagtgta aaagctttcc 23220 tatttctcca catcctctcc agcatctgtt gtttcctgac tttttaatgg ttgccattcc 23280 aactgccatg agatggtatc ttattgtgat tttgatttgc atttctctaa tgactagtga 23340 tgatgagcat tttttcatgt ctgtcggctg cataaatgtc tttttttgag aagtgtctgc 23400 tcatatcctt tgcccaattt atgaggttgt tttttccttg taaatttaag ttccttctag 23460 attctggata ttagtccttt gtcagatgga tagattgcaa aaattttctt ccattctgta 23520 ggttgcctgt tccctctgat gatagtttct tttgctgtgc agaagttctt tagtttaatt 23580 agatcccatt tgtcaatttt ggcttttgtt gccattgctt ttggtgtttt agacatgaag 23640 tctctgccta cacctatctc ctgaatagtg ttgcctaggt tttcttctag gatttttatg 23700 gtttgcagtc ttacatttaa gtctttaatc catcttgagt taatttttgt ataaggtgtt 23760 aggaaggggt ccagtttcag ttttctgctt atggctagcc agttttccca acaccattta 23820 ttaaacaggg aatcctttcc ccattgcttg tttgagtcaa gtttgtcaaa gatcagatgg 23880 ttgtatatgt gtggtgttat ttctgaggtc tcttttctgt ccattggtct atatatccgt 23940 tttggtacca gtgccatgct gttttggtta ctgcagcctt gtagtatagt ttgaagtcag 24000 gtagtgtgat gcctccagct ttgttctttt tgcttaggat tgtcttggct atgtgggctc 24060 ttttttggtt ccatatgaag tttaaagtag ctttttccaa ttctgtgaag aaagtcagtg 24120 gtagcttgat ggggataaca ttgaatgtat aaattacttt gggcagtatg gccattttca 24180 ctacattgat tcttcctacc catgagcatg gaatgttttt ccatttgttt gtgtcctctc 24240 tgattttctt gagcagtggt ttgtagttct ccttgaaaag gtccctaaaa cttcatttct 24300 aaaaaaaaaa aaaaaaaagg aatatgctac caaataggat tctttaaatc aattgtctcc 24360 tccttcagaa aacatccaga acctgacctc ttatcaccac ctccactgac cactttggtt 24420 taagccactc ttattcccct tcggatgtat tgcagtggtc tcctgacagt ctcatttcta 24480 tccttacctt cctagattcc agaggttgag agtctgccct cgacttagaa gccattgtga 24540 tactgttaaa atataagtca gatcatgtta gccgtctgct ccagtgtctc cagtggtacc 24600 atatctttta gagaaaaatc aaatttctta cagtggcctt caaggcccta cagcatctgc 24660 actctgctgc actctgattg tactatctac tactcttctc cctggcttag ccagctctag 24720 ccttactcac atcctcaaac aatccctgtc atctcttagc atcttagcaa tttcttcttt 24780 cctctctaat tcctttcccc aacactcccc acttctccaa cccattccag cttccagctt 24840 ccagcttcca tgagtatcct gaaacataac aaactttggt tactgccagt gtctcactta 24900 acacattccc ccactacagt gttgtcaagt tgtttcattt aacaccagta acatttaata 24960 tcagtaatag ccagattcta tcttaggaaa aatatgccta aaatttattt gctgaaaaaa 25020 aaagaaaaga aaaatttgga taataatctg ctttgagtta ttacaaacac agcagtatct 25080 gagaagcaag actgagtgtc ataaggaaac aaccagatta aataaaagca tagctggatg 25140 agataagaaa atactgcagt gtgatccaaa atgcagtaac agagttaaaa tccatattgg 25200 tcgtcataaa gagcaggaaa taacaccaat tatgtagagt tcacatttaa ggagttactt 25260 cggaatgaag agaaattggt aaactggtga aaattacaca ctatgattta ttgtttttat 25320 tatgtaccag tcattgtggt aacatttcat ctaatttagc tcacttaatc catataacag 25380 ccttataaaa tgtttatctc tatattatat gtgggaaaaa ctaagatttt gattaaacaa 25440 ttttatcatg ctgcagaaag tgacagaact gaatttgaat gaaagtctat ttgactctaa 25500 agcatatggc cttatcacta tgctgttgga caacagatac gatgaataga gagcacatat 25560 gcaacccata aatatccagt gattctgaag aagaaactaa aaaacaaggc agcaaaagta 25620 ttaattgatt atagaaaatg actcttcctt atgtatcttt tttacgagat catttctaca 25680 ttgtgtcacc agcacctaga acaatgttgg cacatagtag atattcaaga aatatttgtt 25740 tattgaactg tcataattac tgttaagcat ttattctgtg tctgttatgg tgctatagat 25800 tttatataca ttattttacc ctcaaagcaa cttttaaact gttattttat agatgaggat 25860 aatgtagtgc caagaagtta aatgatctgc tcaaggttat atagctaata ataggtggta 25920 gaacacgtat aacagaagta agtttagcag ttaaataaaa attttacttt ttagatcaaa 25980 aggactcact gcatcccagg taaatgtaat tttaaaaagc taaaactttg caatattggt 26040 aattttttaa aatttttcat gagaaggaaa aacaccctat atgcatttaa gtaggattaa 26100 taaaactggt taactactaa gacataaaaa ttgggttgac attagatttc ctctttgagt 26160 actaaatgct gagattggca aaaattccat caggttgagt tgctattctt gggtgaagat 26220 aacatatatt cttatatatg tgagaggtca ggaatatgcc cagtctttac cgaagtactt 26280 gtttgtctga tggttggact aatttgggat tcctgtagtg tcctgtagtt cagggtgaag 26340 tttagtaaca tgcgggggta agagacagga ggtgctattc tctgccctgc tgctcaccag 26400 tgtgctgtat ccctcctcag gatcaccctt aaggtctcag atgctcacac ctggtgttca 26460 gtgcccagtg gtctgttctc tagccagtat attctgtgtc gtcttagatt ccagtccaaa 26520 gactatttag atctgctcca tccttgggaa tttccacgca ctttattatt tcttctaggg 26580 caggggtccc caacctagaa caagcctacg atttgtgggt cataccatac tgactttcac 26640 catttcactc cttgaactat tgaacataca ttatcatttc tcctgccatt ttactcacct 26700 gctgctcacc tcctgctgtg aatcaggcca cacagcagga ggtgagcagc aggtgagtga 26760 gcattactgc ctgagctgca cctcccatca gaccagtggt ggcattagat tctcatagca 26820 gcgcaaaccc tattgcaaac tgcgcgtgcc agggatctag gttgtgctcc ttatgagaat 26880 ctaactaatg cctgataatc tgaagtggaa caatttcatc ctgaaatcac cgcttccccc 26940 aacccccccc acccctcact gcccccagtc cgtggaaaat tcgtctttca tgaaacctgt 27000 ccctggtgca aaaaagatta gggactgctg ttctagagaa attaaatgtt tttatgtcta 27060 ctttgttaca aggttacatg aaataaacca gttaaatgct tagtatattg gtctctaata 27120 tactcaataa gattttcctt ctctccttca ccccacacct ttttccttca cttttatcaa 27180 aaatccttgg atcttcatga agatatttta aaacctactg atttgttatt gtccagctgt 27240 ctttcacact ctgctagata gtcactcttc attccacttt acatgaccat cccctgactt 27300 actaacgtga aaaactggtc tgaacctttg tctcattctg catgcatctt cagaatatgc 27360 tggattccag aaaacataca aaaaacttta aggaatagta cagcgaatat ctgtaatcaa 27420 caatttgaag gtaagttgca gacatcatga catttcacct cttaaggctc caaaattcat 27480 gtcctaagga caagaacatc ttccattaca accacaatat tatcacccca aaattttaac 27540 attgataaaa caattctaat attcagctag tattaaaatt tcttcagttt ttcccaaaat 27600 gttttgtata gaatgttttt ttattcagga tccagtcagg aattgtgtat tttacttggt 27660 tagcaactct gtttagtttt ctttaatcta aaaagatctc ccaccttttt ttgtctttca 27720 ggacattggc atttttttgg gagtctaggc cagccgtttt gtagaaagtt ccataatctg 27780 gctttgtctg atttttcccc cacataatta tatgccagtt aaacgttttg acaagaatcc 27840 tacacaggcc aacggttctc agacactgat ctcatgaccc ccttatgtgc ttaaaaattg 27900 ctacaagccc aaagagcttc tgtatacgtg aattctctct ctcagtattt actgtgttag 27960 aaattaaaac tcataatgtt ttaaaacttt ttattagctc atttaaaaaa cagcaaaccc 28020 attagatact aatttttcaa accaaaaaat ttaatgacaa gaatgacatt gttttacatt 28080 tttataagtc attttaatgt ctatcttaat agaagacagc tggattctca tatctggatc 28140 tacagtcaat ctgttccaat atgttgtttt ggtggaagca tgggaaggaa atgtggcctc 28200 acacagatat gtagttggaa aatggaggag tattttaatt gtcttttcag acttgacaag 28260 tggtggccat ttcattagga attgcaaaag gatgattctc tatttctatt atcccttctg 28320 attttatcag ctataaatat ttttaatagt tctctctctc ttccaagagg caatctggta 28380 atcttaaaat acacttaata atggaaagcc taacagatac ttattattta cttttaattt 28440 tcagttttca aagttgttaa gtttgtgccc tagtaatctc cagtggtttc cagtgagttc 28500 ctcttgtttc tttcattctt tctcttctta catatcatta tgtactcatg ttttttaaaa 28560 tatatattca atgtgtttta attacttgaa ccagttcttt ttgatgttca agtggtttta 28620 attttggttg gtgtgaatta tcagtggtca atgaaacaca attttattag ccctcagtaa 28680 aagcaaatca tctgtcacag caaagcatcc caggctcaac ttgcacattt cctgccctag 28740 tcctggatca gtcatttccc caaggagctc tggttcgttt tagatgaaaa ttatattaca 28800 tgtgaaaatc tgaattcctg ctggagttgt ctttactttt gggcattttc agtgaagaga 28860 cctagaaaat acctcttttt gaaaaagtaa ggggatcatg agtttatacc atgatttctc 28920 aacctcacac tattgaccta ttgggccaca taattgttgc agggggggat gtcctgtgca 28980 ttatgggatg ttattagcct ttttggcctc tctgcaataa tacctaccct ctagttgcat 29040 aaaccaaaat tgtctccaga tattgtgaaa tgtcctggtg aggaagggag caaaattgcc 29100 tctagttgag aattactggt ttatgtttgt atttctagca aaatcaaaat aattactagt 29160 tgttttatct gtatgtattg tgtgtgtgtg tgtgtgtgtg tgtgtgtgga ttcaaaatac 29220 tagtatcagc attattacct ctaacaacaa aacctgcttt gtagactagt tcttccatct 29280 cctgcataaa atcttaggcc agtcccaaag attttaatca gaagaacaat ctcattccac 29340 taaccgtatt agtctgttct catgctgctg tgaagaaata cctaagactg ggtaatttat 29400 aaaagaaaga ggtttaattg actcacagtt ccacatggct gggaaggcct caggaaactt 29460 acaatcatgg cccaagggga agtaaacatg tccttcttta caaggcagca ggagagagaa 29520 tgagtgccag taggggaaat gctagactct tataaaacca tcagatctca tgagaattca 29580 ctgattgatc acactgctca tgatgatcac gagaagagca tggggggaaa ccacccccat 29640 gattcaatta cctcccactg agttcctccc acaacatggg gggattatgg gattacaatt 29700 caagatgagg tttgggtggt gacacaaagc caaaccatat cagtaatcaa aatgcccagg 29760 gaagcattat gccaaattta caaatcctct tctaagttta gatttcttct ttgccccaat 29820 tcagcactca ctgctttgag cttgaagcct aaagggaaga acaaataaga tctgcttctt 29880 ctctgtttat gctgcctaca ctcacccaat gacagctgat tttgaccctt ccagggccaa 29940 cgtgtgggaa gagaagttag agagcacagt tttacacaac aagtaggaat tttagtgtca 30000 ctggggttat ctgtgtttgg tgggtgatta caggctgctc ttcctctagt gggatacttt 30060 ggtgagattc atggagatac tttactgaga gcctctaact gtaatcctgt aatgctgtaa 30120 tgcagtagac acctctcttc tctctggaca tgaccgtgac cctctgcttc tggacttccg 30180 ctgactgacc ctacacagat tctgttggat cacaatactc tctaggttgt cctcttagct 30240 ggagccagtt tttaaaagaa agaaactttt attgataaat attttcagaa aagcactcaa 30300 agatgtagag cttatcagta ggtaaatgaa acgatgttat tatcacccag accaaaaaat 30360 ttaaagatca ttaggttccc agaagctcca ttttgcccct tccaatcatt ccccaaaagt 30420 aaccgccatc ccaatttcta acatgataga ttggtttgaa ctattttaga actttatatt 30480 aatggaatca gatagtattt ttctatgtct gcttatttta cacaacatta tacttatgta 30540 gttcatccac attttgcatg gagctagaca tttattctca ttgctgtata ataatgcatt 30600 gaataaataa aaccatttgt ttacacatta tgctgtaggt gatcatttgg gttgttttca 30660 gattgaggct attatgaaca ctgctgctat aaatattctt gtacaaatct tttatttttt 30720 gcatgtggac acacctgtcc ctacagataa gcttttcatt tgcctctcca aggaccctgg 30780 caatttcact ggtcctgtac cagtctttgt tccttggccc aggttcctat accatgtgat 30840 agtgaaaatt cacctcccat taaaggccaa tattttattt tgtttatgtt gtttttggca 30900 taaatgattt ttgtcctcac cctgaatttt ggacagattc ctggctgcct ggcattttct 30960 gtgtagtgtt ttctagccct gttttcattg ctaaggcagt cttttaaggt tctacatttt 31020 tatgcatata gttcagccct gccactctct tccattggga ctgaagtaat atatcccatc 31080 ctctcatggg ctttaaaccc catttcttat cttttaggca tttcttatat ctgtatctga 31140 aacttttttg gatcactgca gagtccagtt aagtttgttc ctccagcttt catttctttt 31200 ttcttttctg gcttatagag atttacctac ctttttttcc agactgaaat taagcaacat 31260 gcttctatta tattttatcc agtatttctg tgtttgtggt agaaagattg ccctcatatg 31320 gaccagctca tgttgcttca agcccgtgac ttgtgggtca taccatactg actttcacta 31380 tttcattcct taaactactg aacatatatt atttcctgcc atttgactat gtattttccc 31440 cttcttttca aaaatgccat gaaaaattgt gaactctctc agacctaagt gaactatgtt 31500 cacttgtgct catattgaga agcaacacaa acaatatagg tctcttattt tacaggtaag 31560 tccctttctt gtttctaata aagatatttt atttcctaat aaagatattt tatttccttt 31620 aaagtggaat tttgtcatct aaaatttttg taagtcacta ggcaaataaa atgatctaat 31680 cacattttga tagtgctttt attttcacag agtatttttt attaagagta acacttgggg 31740 ctgggcacgg tggctcacgc ctgtaatctc agcacttcag gaggccgagg caggaggatc 31800 acaaggtcag gagatcgaga ccatcctggc taacacggtg aaaccccatc tctactaaaa 31860 atacaaaaaa ttagccagac gtggtggtgg gcgcctgtag ttccagctac tcgggaggct 31920 gaggcagggg aatggcatga atccaagagg cagagcttgc agtgagctga gatggctcca 31980 ccgcactcca gcctgagcga cggagcgaga ctccgctcaa aaaaaaaaaa aaaaagagta 32040 acacttggaa ataatcttgt tagacttcac atttaattat ggtctattac tataaaaagg 32100 tgagaaatta ctgaataaaa agcattctgt aagaaaaaaa gttaaaaggt ggtaaaaaat 32160 cagaagcaag tgactattta ctatatatga caataagtga catatgtctt atactgttga 32220 gtataactat atttacaaat ataagcgaaa caataggtat aattgtatat acacctatct 32280 aaaaaatatt tatcaagctg tattttacac aagagttaaa agtatcaaac tgagtagttc 32340 ctactcttat agaacctagt agctatagac acaaagtata caataggaat acattgaatg 32400 tgcagttggt acacagaaga aatgtctaaa gcctttatga gagcagtcag gtaagaccag 32460 atgaggggag cagttaagcc aaggcttgaa agacgagtga gattttgtta agttgataaa 32520 ttggaaggaa gggaggaaag tcatagaggc atgagagagt ttgggggtaa tccaaggatg 32580 ttagtactct ggagcataaa gaacatggga aagagtatgt gattagaaag aaaagacata 32640 acagtttgaa ctcaccccct gctttcccca ccctaaaata gatcagtttt gcaaggagaa 32700 agaagcatag tgctctatta agctccccag gtttcaacag gggcagatga tgcatatatt 32760 aagggagaga gctagatagt ggctcctagt ggggtgaact cccctattta tcattatgcc 32820 atgtaacctg tttgtggtgg cttatagctt ataagtgttc ctaaatgtct tataactcct 32880 ggtggtgata atgtaggtaa acagcaaaga tggcagcaaa tggaatgctc tgagtggttt 32940 aatatagtga cgtgttttac acttgttgta gaatcacaat gttatgttgg actgaggaga 33000 agaaataata cgcaatttga gccatcaacc tacggtttat gagtgagtga gtggatctag 33060 acagggatgt gtttctcaaa accagaatat aaactgtggg ctgttgtctg aagaaaatgg 33120 tagcaactga tgttatgtta gattttcatc tttttctaga tttccaggaa aatgttgtga 33180 cagggctaat gcacttgtgc ctgacctttc ttcacctgca tatacagcca ggtacctgaa 33240 gtagcgagtg gctcccactg cattcattgc attcttggtg gctctgagtc ctggccatgc 33300 attatggttc agaggcaatg cctgggcccc atgccaaatt cccttagtcg gctctagttg 33360 gaactggctg gctacaacat ttctttaagc caaaacattg cttgagaaga taaaccttta 33420 actgtactcc ccatattttg tttgcatgat aatgcatcta ctttgcagtt ttgatggctc 33480 atcacttaat gttaaagatg atgaaaagtt agagtgaatt gagcttccag atttctttaa 33540 gccattccag cctgtcacag gcagtctaca atgatatgag tcgtgccaac ccctcaaggt 33600 cgtgacttca tgccagtgcc cttaattatt aaaaattatc cttccaactc aaactttaaa 33660 tattggacat ctaagtcatt ttttcttcca aacttataag aattgcattc tgtttggtaa 33720 ctataatttc cttatttata ctcatgttta aatttatact tatgtttaaa tttatttggt 33780 acttatcatc agtctcttca taccagactt cctacctcat acattgtctg ctataacgta 33840 tctgttgttg actggattac atcttttgga agagcatact ggggtcttat ttcccaattt 33900 catgcaagtt tgagaatatc tttaagttgc ctttaaaact gaataacttg actagatata 33960 gacttcttgg accacatcta gaaaaaatct aactatattt gagatttaga tgtgtgataa 34020 tcataattat aatagacatt tgttaagtac ttgctatgtg ccagactatt ctaagctctt 34080 acatgagctt gtgtattcct tagatccaat ccaaggtact aatgttatct tcattttaat 34140 tgccaaatgt catctagcta ctaagtgatg agattgacat tcacacttaa acagtaactt 34200 tctatactac ctctcaagat aattaaccta aaatgtttta actgtgtgac cttgagcatg 34260 ttatttaact tctagatgcc tcagttaact ctgtataaaa tgagaatatt aatacctcca 34320 aaggttgctt tgatgatgaa atgagttaat atatgtcaat agctcataac aatgcctggc 34380 acatagtaag tgcttaataa ttgtctgttg ttcttttatt atttttattt gtggatagta 34440 agttgcagat ctgagctttg aacaagcagt ctggcctctg taactttgct cttcggtact 34500 acaatgtagt gtcagcaatt aagatggcat tttaatttag tgggggaaaa catggattag 34560 tcagtaaata gtgttgggat atagtagata ctcaatactt gttgaatgaa tgagagtttt 34620 ataaaagtat aaatacatta acttaaatat taacaaatag atattaaata ccatttagat 34680 ttgtatagac agtttggaaa tactttagtc tgttccactg caataaaaaa ataccttaga 34740 ctgggtagtt tataaataca gaaatatttt ctgatggttc taaaggctga gaagtccaag 34800 atcaaggtgg cacagattcc atgtctggta aaagcttgct gtcttcttcc aagatggcgc 34860 cccttgctgc atcctcacat ggcagaaagg ataaaaagga ccaaattcac tccctcaagc 34920 ccttttaaaa gggtactgat tccatccatg gaggcagaga ccccacctct taatattgtc 34980 acattgggga ttaagtttca atatgaattt tgaaggggac acaaacattc aaaccatagc 35040 aagaaggaag caaaaatgag aatagatgca tgattatcaa attgtaggtt attttctgtt 35100 ttgaatttcc ttacgttctt atataatttc tctaataata caattcatga tataatttgt 35160 acagataacc acttagttgt attttgtata tattgagttt gaggtaccta gaaatatgac 35220 tggaaggtca gagctcatat tgagattttg ggaccatcag aagataatga gacagactac 35280 ccacaaagtg tgtgcagtgg cacaccacat ataaggaata agcaaagtaa gaggagttga 35340 caaagggaat ttaaaaggaa gtgctggaaa gacaggtcaa gagtcagaaa agagaggccc 35400 caaaatatga atcaatggga caggtgggga aagaaattac ctgaagcaaa tgattcaggt 35460 caggaaagca ttcacagcat ttaggaatga aaaaatattt gatgatgtta agccatttta 35520 taagaatagt gaatatacaa gtcaaattac cacataggaa taaaggaagt taggtagaaa 35580 cagcaaatgt agatgatgtt ttcgacaact ttgaggacca aaaaaagggg aatgtattca 35640 gggagaatct agagtgagct agggttcctt tttttggtgc tggaactttt ttggatggaa 35700 aaataatgag agagcaggac atcactggac tggagagact aaagacttta ggagagaaag 35760 gaggaaattg atggagcaag acttgaaagc agataggaag gaattgtttg aaggacaaaa 35820 gcaaaaacca ttggtaagga ggactctttg agacactgaa taaggagatg atatcgtagg 35880 ccattttaga aatgtaagtg agacagatca gcagcatgcc agcttctggt gtttagatca 35940 gacctttttc ctgcgtattt atgaccaagg agcagacagt aggacagcct ctcttcaaac 36000 aattacatct gcgtgctcta tttctcgaag tctgttttct gtatagagaa aaccttggtg 36060 ggcgtggtgg ctcatgcctg taatcccagc actttgggag gccaaggcgg gcagatcacc 36120 tgaggttggg agttcgagac cagcctgacc aacatgcaga aaccctgtct ctactaaaaa 36180 tacaaaatta cccaggcgtg gtggtgcatg cctgtaattc cagctactca gaggctgagg 36240 caggaaagtt ccttgaaccc aggaggtgga cgttgtggtg agccgagact gtgcctttgc 36300 tttccaggct gggcaacaag agcgaaactc cgtctgaaag aaaaagaata gaaaatcttg 36360 ctattttctg ttctctcttc ttgtgagata tcagtatagg gtagaaatag atgatcaact 36420 gatagactga cttacacaca ccttgctctc acagtcactc ctatagccta aatatataac 36480 ttgtggtaaa aaaaaaaaaa attagagtca aatcagaaag tagtatgcag tacactctgc 36540 taacctcctg taggactggc actatgtgca caaaagtcag caaagatatg aggcttcaca 36600 gttacccttt attaggtgaa ggcttaattc ctgggactct aaacaagaga tgaacactat 36660 gtgagtgtac atgaacataa gtttatagag gcatgcatct ttaagaaaat atttgtgctt 36720 ttccagaaat gcatgaggtc aggaaccttc ttgatatcag ctgtcacata tataggcctt 36780 ttggcatttt tgccctttgc cgtttaggct aatcctttcc cctgcttgat taagtacatg 36840 ttttaggtac ctctgagttc ctttactacc aactttggcc agaagactct gattttaatc 36900 ttaactaatg taaaatacta caggaaaatc ccatgatctt ttaaaattat gtgtcatgct 36960 tcagcagtat aagtaggctg aagtaggctg atgatattaa aagactcaca gtctgtttta 37020 aagaaattta ttgggaaaaa aagagagtgt caaaaatagt aaattaacgt ttacatgttt 37080 aagctgaatt tttttatttt tgttaaatct aaataaatta gattgttccg tgccttatta 37140 acatatttgt ggtttaaaaa tcattattga atattaaatt catgtgccag acctttttaa 37200 aggcatccat agtttcctct acacaaagtg accccagggt gatgtgcgat gtgtattgtc 37260 tatacttcta taaagtttct cctctttatt gagtacttct atctgtttta agttcttcct 37320 ttatcattat ttctgttcaa ggtattttct aatgtgtagt caaaataagg caggtattaa 37380 attcagtaca ataagtgatc ttttttacat gctcttccat tgattcctta aagagagaac 37440 cagctctggg atttttccaa tagaactaag catttagatt tttttttaaa aggtcttttc 37500 cagaggcctc ttcaattctt tagtctgtat ctgttttctt gaaggcatca atgtcagaaa 37560 gaaaggtttt actagagttt gaccacctgg tacaactaat gcttatcaga aacgtgtagc 37620 taatcttgaa atgacatcta aaggcaatct gatactgaca ccattaagaa gataaacaga 37680 aatctcttgc aaaagatttt taaaggagcc ctaggattga ttcttcagct atgctcacct 37740 aatttaggct cctaaaggca caggtaaagt actgagcagt tttatagctt caccaacatt 37800 cagctgtgcc aggcaggaag ctggctgagt catgcacttg ctgatgtgta tctagtctct 37860 atttcagttg tcactgcata gaatttccag tgaatcttag atattgtaaa taattttgac 37920 tcattttgtt tttttagggc agcacaacta cttatgtgct ggaagaaatg actgcatcgt 37980 tgataaaatc cgcagaaaaa actgcccagc atgtcgcctt agaaagtgct gtcaggctgg 38040 catggtcctt ggaggtaatt ctgatgtttt catcaataat atactgtgta atctttatac 38100 tataaaactg tgtgtcagaa aattgcaatt tcttattcat ctttgttttt gttttttctg 38160 tcttgattat tggcagtgac tctgcacatg tgagtgtctg aatgaagcaa ccaataccat 38220 tccattaact ttacagtttt ctagcacatt gaggtctgtt aagagaagaa aactcaattc 38280 atctatattg cctataagta gaggtaatgg gcaatgtact gacctgtcat taagtaacat 38340 gtgtgggagt ctaggctttg ctgcccatct cctgtgtgac cacagctctt cctgggcatc 38400 agtctactta tctgtaaaac gagagcatta acattccttt cagctcaaaa aattgactct 38460 gcaaattggt caggtacatt gcttactagc ttctccaagt gattgattaa agcaagatta 38520 ctgtaactgg ggtaggaatc catttgatga agagttatat taataattcc cttttcccat 38580 atttttatta gaatctctaa aactttcagt aatatagcac acatttaaaa aattaatgtc 38640 agatagtgac agatagtttg aatgaaaaca tagcagagtg agggagatca agcgagccac 38700 agttatgcaa gacacccgta ctcccttgaa ttttttcact tagccacatc ctgcacaaca 38760 ctgtctggga atcgtagctt ttacatctag atataacttt ttaaaaacat aataaaaata 38820 ttacataatt gttaaagcta aaaatgtttt ttcagtgcac taactataaa cagcttgtgt 38880 tccatggacg ttgtgtgtat tttcctttgg gaaatgctat atttagacca atgcttgtca 38940 agtttaaagc gtatacaact gtcacctcat ttgtttttgc aaatgtgctg gaccaaaatt 39000 aatcacccag gtaacatact gggcacttta cagtaattca tgtaattaga tcacttatag 39060 aattttataa tttcttgatt aaagatttgt tttcctaagt taagggcagt gattaggttg 39120 acagtagtgc ctggcttaag gaggtgctca gtaactttct attaaatgaa taaaaccaca 39180 acctcagcag attaaaattg tttttgagtc ccacacctca attaccacct ctttgtattt 39240 ctgtttcaac tcagaagtct taaggaacct cctaaatttt agaaaataca actttttcac 39300 aaaatcacac atctagtcaa tgtcggagcc agaagaaaga cctgaaggta ttctgaccct 39360 ggcgcagcca ttgttttctc cttggcatgc ttgccttaga aagtagcaga gcatctaatg 39420 ctgacactta gattccatta agatctttaa tcttgagtgt tttcccaatg actttaaaag 39480 actcttcaat tttatcaaag aatgaaaatg gcggagttct ggatagaacc ttcagaatac 39540 tcctggggat cagcaatata acctagagaa atttatgtta tcttttaata ttctagggct 39600 ttatttattt atttttgaga cagagtctca ctctgtcgcc caggctggag tgcaatggtg 39660 caatcttggc tcactgcaac ctctgcctcc caggttccag cgattctcct gccacaacct 39720 cccaagtagc tggtatcata ggcacccgcc attacgccca gctaattttt gtattttagt 39780 agagatgggg tttcaccatg ttggccaggc tggttttgaa ctcctgacct caggtgatct 39840 gcccacctcg gccttctaaa gtgctgggat tacaggcgtg agccactgca cccggccttt 39900 agcatattat tcaagcatct gtaatccttt atcttctcta accaaactgc ttgttacttt 39960 agcctgttat tcctatgctt cccaagccat attctacggg cttcacagct attgctgcat 40020 tttcatacct tttatattac ttatttgttt aactctactc cctccatctc tccctccttt 40080 ccgtccctcc ctccctccct tccttcttct ttccttcctc cctcttctcc ctcaatcgct 40140 ttctaatttg caccccatcc ctttctttca gtaactgtat atcgagccct ttctagttac 40200 cagatactat tctaagttct gcgcatatag cagcaaagaa aatggacaaa attcctgccc 40260 tcatagatct tacactgtgg gaggacgagc aacaaatgta catgtcaaat aatgataagt 40320 gctgtggaaa gatattaagc agagttagtg gggattatca ggaagcagac tcactactaa 40380 actcattagt ctaatatttt tgttttgctt agcctcttcc taagcaataa tatctctgaa 40440 ataatggatt tgatatgcca gttataattt ttctaaaaga cattaatata cattcttaac 40500 tatttaaata tatattcgat gtaccaccta aaaactgctt gtataccata aatgattatg 40560 tgttaaactc tgggaaagag tgtattagac catttttttc cagctatcgc agctctattt 40620 attttttcag cttttatttt agcttcagtg ggtacatgtg caggtttgtt acctgggtat 40680 attgtgtgac actaatgttt gaggtacaaa tgatcccatc acctaggtac tgaggtactg 40740 agcatagtac tcaatagttt ttcaaccctt tcttcttctt ccttcctcct ctagtagtcc 40800 tcagtttcca ttgttatgct catgaatacc caatgtctag ctcccactta taagtgagaa 40860 catgcagtat ttggtttttt gttcctgtgt taatttgctt agggtcttgg cctacagcta 40920 catccatgct gctgcaaagg atgtgacttc gttcttttca tggctgcata atattccatg 40980 gtgtatatat gccacatttt ctttgtccaa tccactatta atgggtgcct cagttaattc 41040 catgtctttg cttttgtgag tagtgctgtg atgaacatgt gagtgcatgt gtctttctag 41100 tagaacaatt tattttcttt tggctatata tccagtaatg ggattgctgg tctaatggta 41160 gttctgtttt aagttctttg agggccggga gcggtggctc atgcctgtaa tcccagcact 41220 ttgggaggcc aacgcaggtg gatcatgagt tcaggagatg gagaccatcc tggctaacac 41280 agtgaaaccc cgtctctact aaaaacacaa aaaaattagc caggcatggt ggcaggcgcc 41340 tgtagtccca cctacttggg aggctgaggc aggagaatgg catgaacctg ggaggcggag 41400 cttgcagtga gctgagttcg cgccactgca ctccagcctg ggcgacagag cgagactgtc 41460 tcaaaagaaa aaaaaatgtc ttttgagaag tgttcaaacc aatttctacc gtggcagaac 41520 taatttacat tcccaccaac agtatataag tattcccttt tttccacagc ctcaccagca 41580 tctattaggt tttgagtttt ttgagttttc ttttttgaga cagagtctca ctctgtcacc 41640 cagactggag tgctgtggca caattatggc tcactgcagc ctcaatctcc tgggctcaag 41700 tgatcttcct acctcagcat cctgagtagc tgagactaca ggcacgcacc accacacctg 41760 gcaaactttt tatttatttt ttttttgtaa cgataaggtc tcactttgtt acccagtcag 41820 gtctcaaact tctgggctca agtgatcctc ccaaagtgct ggaattacag gtgtgagcca 41880 ccatgcctgg catgagtttt taataatagt cattctgatt ggtgtaagat ggtatgtcat 41940 tgtcgctttg atttgcatat cttaatgatt agtgatatgg agcattttaa atgtttgttg 42000 gctgcttgta tgtctccttt tgagaaatgt ctgttcaagt cttttgccca tttttttggt 42060 gagatttttt ttttttgctt gttcaactgt ttaagttcct tatagattct ggatattaga 42120 catttgttgg aggcatagtt tgtgaacatg ttctcctatt ctgttggttg tctgttaact 42180 ccattggtag tttattttgc tgtgcagaaa ctctttagtt gaattaggtc tcacttgtta 42240 atttttgttt ttgtttcaat tgcttttgag aacttagtta gaaattattt cccaaggctg 42300 atatccagaa tcatatttcc tagattttct tttaggattc ctatggtttg aggtgttata 42360 tttaaatctt taatctacct tgaggtaatt tttgtatatg gtgaaatgta ggggtccaat 42420 ttcattattc tgcatgtgac taggtaggta tcccagcacc atttattgaa taaggaatcc 42480 tttctgcatt gcttattttt gtcaacattg tcaaagatca gatggctgta ggtgtgcacc 42540 tttatttctg ggttctatat tctgttccat tggtctatgt gtctgctctc gtgccagtat 42600 catgctgttt tggttactgt agccttacag tatagtttgg aattgggtaa tgtgatgcct 42660 ctgacttttc tttttgttta ggactgctgt agttatttgg gctcttttga ttttatataa 42720 attttagaat agctttttct agttctggaa aaaatgtcgt tggtagtttg ataggaataa 42780 cattgaatct gtaaattgct ttggacagta tggccatttt aacaatactg attcttctaa 42840 tccatgaaca tggaatgttt ttacatgtgt tagtgtcatc tgtgatttct tttagcaatg 42900 ttatgtagtt atccatgtag aaatctttca tctgcttagt tatatgtata cctaggtatt 42960 ttgtgtgtgt gtgtggctat tttaaatggg attactttct tgatttggct ctcaccttga 43020 atgctatagt tgtatagaaa tggtactaat ttttacatgt tcattttgtg tcctgaaact 43080 ttactgaagt catttatcag ttttaggagc cttttggtgg agtctttagg gttttctagt 43140 tataaaatca tgtaatatgc aagagagata gtttgacttc tttttttcat gtttgaatgc 43200 cttttatttc cttctcttgc ctgactgctc tggctagcac ttccagtgtt aacaggagtg 43260 gtgagagtga gcatccttaa ctttttccag atctcaagga aaattcttcc agcttttgcc 43320 cattcagtct gatattggtt gtgagtttgt cattttggtc tcttattgtt tttaggtata 43380 tttctttgtt gcctagtttc ttgagggttt ttatcatgaa gtggtattgc attttatcga 43440 aagctgtttc tgtgtctatt gagatgatca tatggtttta tttttaattc ttctcatgtg 43500 gtaaatcata tttattgatt tgcatatatt gagctaacct tccataccag gagtgaagcc 43560 aacttgatcg tggtaaatta actttgatgt ttgatttggt ttgctagtgt tttgttgaga 43620 atttttgtgt ctgtgtttat cagagatatt gtcctatagt tttcctttct catggtatct 43680 cttacaggtt ttggtatcag ggtgatactg gcttcagaaa acgagttagg gaggagtctt 43740 tccttctcga ttttttgaaa tagtttcagt agaattggta ccagctctac tttgtgcatc 43800 tggtagaatt tagctgtgaa tctctctggc cagggctttc tttttggttg gtaagttttt 43860 tattactaat tccatttgga acccaatatt ggtctgttca gtgctttagt tttttcctga 43920 tttaaacttg ggacattgtg tgttttcagg aatttattca tttcctctag atattgtagt 43980 ttgtgtgtgt agaggtgttc acaatagtta tctaaggatc tttcatattt ctgtgggatc 44040 agttgtgatg tcacctttgt catttctgtt tgtgtttatt tggatcttct ctcttttttt 44100 ctttgttaat gtagctagca gtctatcaat cttatttatc ctttaaaaaa ataacttttg 44160 gttttgttga ttctttgtat agatttttgg gtctcaattt catttggttc tgctctttag 44220 ttcgtccttt tcttctgcta gctttaggga tagtttgttc ttgttttttt tttttttttt 44280 ttttttttag ttcctctagg tgtgatgtta ggtcattaat ttgagatctt tctaactttt 44340 tgaggtaggc atttagagct gtaaactctc ctcttaatat tgtttttgct acattccaga 44400 gattttggta tgttgtgtct gttttcattt attttaattt ttttttattt ctgccttgat 44460 tttattgttt actcaaagtt atccagagca aactttttaa tttccatgta attgtgtggt 44520 ttgtacagat cttcttgatg ttggtttctc tttgtattcc actgtgacct gacagtatga 44580 ctggcatgat tttgactttt taaaatgtac tgagtcttgg tttatggcca aggatgtggt 44640 gaatcttgga gtatatgttc tgtgtacaga tgagaagaat ttacgttctg tggttgatgg 44700 gtggattatt ctgtagatgt ttattagatc caattggaca agcattgagt ttaagtccag 44760 aatttctttg ttagtcttct gccttgatga tctaatgcta tcagtccccc actattgttg 44820 tgtggctttc taggtcctta gaagtccttg ttttatgaat ctgggtgtgc cagtgttgag 44880 ggcgtatgta tttaggatag ttaaatcttc ttattgaact ctttatcgtt atgcccttat 44940 ttttcctttt ttactgttgt taaagtctgt tttatctaat ctaagaatag caacccctgc 45000 tcttttttgt tttctgtttg catgctacat ctttctccaa ccctttactt tgagcctatg 45060 gttgtcatta catgtgagat gggtttcttg aatacagaag atggatgagt ctggttttcc 45120 tagctggttt gccactctgt gacttttttt tttttttaga tggagtctta ctctgatgcc 45180 caggctggag tgcagtggcg caatctcagc tcactgcaac ctccacctcc caggttcaag 45240 caattctcct gtctcagcct ccggagtagc tgggattata tgcacctgcc accacggctg 45300 gctaattttt gtatttttag tagagatggg gttttacctt gttggtcagg ctggtcttga 45360 actcctggtc tcaggtgatc cacccgcctc agcctcccaa agttctggga ttacaggtgt 45420 gagccactgt gcctggaccc actctgtgac ttttaaatgg gacattgtat tagtccattc 45480 ttgtgctgct ataaagtact acctgagact gggtaattta tgaagaaatg acatttaaat 45540 gactcatagt tctacaagct taacaggaag catagctagg caacgtcagg aaacttacaa 45600 tcaaggcaga aggtgaaggg gaagcaagaa tatcttacca tggcagagta ggagaggtgg 45660 gtcaggggag tgccacacac ttttaaatca ttatatcttg ggagaaccca ctcactatca 45720 tgacaacagc atgggggaaa tttgcctcca tgatccaata acctcctacc aggttcctcc 45780 ctcaacattg ggaattacaa ttcaacgtga gatttggatg gggatacaga gccaaaccat 45840 atcattctgc ccctggcccc tcccaaagct tatgtccttc tcagttttca aaacacaatc 45900 atgccttccc aacagtcccc caaagtctta actcattcca gcattaaccc aaaagtccaa 45960 gaccaaagtc tcgtctgaga caaggcaagt cccttccacc tatgatcttg taatatcaaa 46020 aacaagttag ttacttccaa gatacaatag gggtatgggc attgggtaaa tactcccatt 46080 ttaaatggga gtaactggcc aaaacaaagg agatacaggc cccaagcaag ttcaaaaccc 46140 agcagggcag tcattaaata ttaaagctcc aaaataatct cttttgtctc catgcctcac 46200 atccagagca tactgatgca agggatgggc tcccaaggcc ttcagcagct ccaccctgtg 46260 gctctacagg atacagcccc cacagctgct tttacaggct ggcattgaat gcctgcaggt 46320 attccaggca cacaatgcaa gctgttgatg gatctacgat tctgaggtct ggttgatggt 46380 ggtcctcttc tcacagctcc actaggcagt gccccagtgg gaactctgtg caggggctcc 46440 aaccccacat ttccctttca cactgtccta gtagaggttc tccatgaggt ctctgcgctt 46500 gaagcagact tctgcctgga catccaggca tttccataca tcctctgaaa tctaggtgga 46560 ggtttccaaa cctcaactct tgccttcttc atacccgcag gcccaatacc atgtggaagc 46620 caccagggct ttgggtttgc atcctctgaa gccctggccc aagctgtacc ttggcccctt 46680 ttagcaacag ctggagctgg agcagctgga atgcagggca ccatgtccca aggctgcaca 46740 gagtagctag gccctgggcc tggccctcaa aaccattaat ccctcttagg cttctgtgcc 46800 tgtgatggga ggggctgctg tgaaggtctc tgaaatgccc tggagacatt ttccccattg 46860 tcttgactgt taacatttga ctcctcttta ctaatgcaaa tttctgcagc aggcttgaat 46920 tgctccccag gaaatgtttt tttgtctctc tcttttctac cacatggcca ggctacaaat 46980 attccaatat attttttgct ttgcttcact tttaaatgta agttccaggt tcagataatc 47040 tcttttttca tgcatatgag catacatgtt tagaaacagc caggtcacat acatctggaa 47100 tgctttgttg cttagaaatt ttttccacca gataccccaa atcatctctc tcaagttcaa 47160 agcttcacag atctctagga caggggcaaa atgccaccag tctttttgct aaagcatagc 47220 aagagtgacc tttactccag ttcccaataa gttcctcact ccatctgaga ccacctaagc 47280 ctagactcat tgtccatgtc actatcagca ttttggtcac aaccattcaa caagtctcca 47340 ggaagttcca aacattccca tatctttctg tcttctgagc cctgcaaact gttccaacct 47400 ctgcccatta ctcagttcca aagtcactcc cacattttca ggtatcttct taactgtgct 47460 ccactctccc agtaccaatt ttctgtatta gctcattctc atactgttat aaagaactac 47520 ctgagactgg gtaatttatg aagaaaagaa ctttaactga ctcacagttc tgcaggctta 47580 acaggaagca tagctaggag gtctcaggaa acttacaatc atggtggaag gcaaatggga 47640 agcaagcacg tcttaccagg gcagagcagc agagagagag agagagagtg agaggggaag 47700 tgccaccact tttaaaccat catatctcct gagagctcac tcattatcac aacaacagta 47760 tgggggaaat gtgcccccat gatccaatca cctcccatca ggtccctccc caaacattaa 47820 gaattaaaat tcaacgtgag atttgggtga ggacacagaa ccaaaccata tcaggcattt 47880 agaccattta cgttcaaggt taatttttat atgtgaggtt ttgatccctt gtttagttgt 47940 tagctgtttg ctttgttgtt tctattgtgt ttttgctttg tagggtgtgc aggctatgtg 48000 cttaagtgtg tttatgtgga agcaggtatg gttcttttgc ttccatgttt agaactccca 48060 tgtaaggatc tcttgtaagg atggtctagt ggtaacaaat tcctttagca cttgattgcc 48120 tggaaaagat tttatttgtc ctgcacttat gaagcatagt ttggcaggat gtgaaattct 48180 tggttgaaat ttcttttaag aacgctgaaa acaggccccc aatctctcct agcttgcaaa 48240 cctctgctga gaaatctgtt attatcctga tagagttccc tttttatgtg atctgcactt 48300 ttctctggct gcctttaaga ttttttctgt aatattgcct ttgaacattc tggtgactat 48360 ataccttggt gatttttttt tttttgagat ggagccttac tgtgtcacac aggctggagt 48420 gcagtggtga gatcttggct cactgcaacc tccgccctcc aagttccatg gattctcctg 48480 cctcaacctc ccgagtagct gggattacag gtgcctgcca ccgtgtctgg ctaatttttt 48540 gtatttttag tagagtcagg gtttcaccgt cttggccagg ctggtcttga actcctcacc 48600 tcatgatcca cctgcctcag cctcccaaag tgctgggatt acagacatga gtcaccacgc 48660 ccagccagtg atgttcatgt ttgtggtgtc tcacagatgt tctctggatt tctagctctc 48720 tagcaagatt aggaaagttt tcttgaatta ttccctcaac tatattttcc agtttgtttt 48780 ctttttctcc tactttctga ggaatgccaa taattcatag ggtttttttg ctttatataa 48840 tcttatattt ctcaaaaaca gctcatttaa aataattttt ttctttattt ttatcagatt 48900 gggttagttc acaagaccag ccttcaagct ttaaaatttt ttcttctgct tgatccagtc 48960 tattgataac gaatttcaaa atataattga aggctttaag tgagtttttc aattccagaa 49020 gctactattg atttttttta agatgttcat gtctttctta atttcctaga ttgctttaga 49080 agtttatgtt ggttttcaac cttgtctttg attacaatga acttccttgc aatccatgct 49140 ttgaattatt tatgtgtcat ttctgagttt ccatcttggt tagggaccat tgctggaaag 49200 ctagtgcaat cccttagtga tgtcactgca atcagatttt tcattatgcc agaattcttg 49260 cactggtttc ttctcatctg gagacattgg cacttcaaat ttttgtactt attttcatgt 49320 gggtagtagt ttttcttttt tttttctttc cctataatgg tattattatt attattttaa 49380 taattttttt tgtttccctt ttaccccttt tctagggctt gtgcctctaa agaatgctgg 49440 gtaggatctt ttcactttga ttctacagcc ctatgtgctt ctttcagcag gttttatact 49500 gggctctgca ggtcatccca caggcccata ggcggcacgt ataggtaaga gctggctgtg 49560 accaatgtgg ctggatatat acttgatcct tgtttccgag caaaagctct ccattgtctt 49620 aggcaatagg ctgattctga ttcatagaat gcacagtagt tggaactccc tgctcagccc 49680 caaggagaag ggaaccacaa agggcaagtt tggaccgagc aggtccacct gcgtcccctg 49740 atggcaagca caagcaccag tgccaaggaa gaatccagtg ggtggccacc aagcacccag 49800 agttgtacac agacctgaag cttggaaacc tcctcagctg caaattctct gcatgggagg 49860 catcctaagc tcctgattca ggagagtggg tgctccagat gcctggagat ctgctttggc 49920 atggaataga gaggggcccc ctgcaccaag atctctacac aggaggggta aatgacaatt 49980 atatatagtg ctgcaggaaa catgtgaatg cagataatat cttcaatata ccagttttct 50040 ttcttttggc tatatgccca acagtaggat tgctgcatca cgtggtagtt ctgtttttag 50100 ttttaggaac ctccatactg ttttccatag ttacagtact aatttagatt actaccaaca 50160 gtgtacgaga atttctcttt ctccatatcc tcaccagcat ttattttttg tctttttagt 50220 aacagccatt ttaactggag tgagataata tcttattgtg gttttatttg catttccctg 50280 atgattagtg atgttgagca ttttttcata tatctgttgg catttgtatg tcttctttca 50340 agaaatttct gttcattttg cctattttta aatgggatta cttttttttt ttgctattga 50400 gctgagttcc ttatatattc tgataattaa ctccttgtca gatgaatagt tttcaaatat 50460 tttctctcat tctttgtctc ttcacattgt taactatttc ctttgctgtg cggaagcttt 50520 ttagcttgat gaaattccat ttgtcaattt ttgccttgat tgcctatgcc ttgaggtctt 50580 actacaaaaa tatttaccca aaagtcttaa agcagttccc caaagttttc ttgtagtagt 50640 tttgtagttt cagatcttac acttaagtct ttaatcgatt ttgattttat tttcgtatgt 50700 ggtgagtgat agtgatctag ttctgttttc tgcatatagg tagccagttt tcccagcagc 50760 atttattgaa gagattgccc tttccccaat gtatatccat ggcacctttg tcaaaaatga 50820 gttgactgtt aatgcatggc tttatttctg ggttctctat tctgttccat tggtctttgt 50880 gtctgttttt ataccaatat catgctgtta tggttaagaa ttctcatttc tgacatagag 50940 accatttaaa taacaaaaga taaaacatga gagaatatta gaaagctttt aaaagattta 51000 ataaaatctt acagtagaat aatagaggta ataaatatgt tttgacatgt catgacatag 51060 attttgttag gagcagtttg ttcttatctg cttccgtggc tatgttttga ttataaaaat 51120 tatgttttga gttatttgat ttcttcgttg catatgattc ttgtctttaa tgaatcactt 51180 ccctaccttg aacaggtcac agtgaacttt tgctttagtt cactgctgta cagcactgag 51240 tgggaagata gttacaccct taaggatgat aatatatctt ctactttagg tgttctgctt 51300 ccatcttctt tcttctagaa aacatttttg tttaatgact cacaaaaaaa tatctgaaat 51360 gaatacagct ttatattttg cagttgtaaa aatgctaaac tagtataagg taattagctc 51420 tttcaaaaat aagtcgcttt ctacataaaa gaatgattaa atgaatagtt atcttctttg 51480 aaatttttta aatccacagt tacaggtttt atgggttttt ctttttttct attattttct 51540 tttttggtag caagccttat cttcacattt taaaaggttg tgttagaaag ccagtttctc 51600 tccttctccc cctacctcct cctccaccta gattcaacca cttcttatgg caaagagaat 51660 aactttctgg gctagagagt atgggttgtg tagtaaagat ttttgttgtt tttttgtttt 51720 ctcctcctac acttgacagc caacaaggct acaagtcttc tttggaagaa ctgcaaaatt 51780 cttgaattta ggacacagtg gtatcttttc tagaagtaca gagtcctgct ctgtgccttt 51840 aataccctaa gaacaaaaat catttttcct ctttaaggca agatctactg tgggtgatgg 51900 ctaccaccta cctctctaga cctcacagaa tcttcttctt aatgggcatc tctttttcca 51960 cccaaatcct ggcctttgaa tcagcatctc tcagagacag cacctctcac attgctcccc 52020 aagttcactg cattggagcc tgcagccaac tgatgtcctt ctcatcagtc tggttgttta 52080 ccctaaacac catctcacac atgctagctg gggatggtcc tcagactatt cattctcatt 52140 tctttgctta aaatttttct gtgtttgtat gggatgtctt ttatggtttg ccaactagaa 52200 cttttcagta gtatctacaa gcatttatta ttttctagac tcagtaaaaa gttagaattg 52260 aggggttcaa atatctcctc cattgtagtt gactttattt gaatgattaa gacatataca 52320 tttaaaaaaa tcaattctag tatagttttg gtttttggaa aatacctgat ttcactttat 52380 atttcctgtt gtctagcatc tttgttttct ttgaagtata tacaaaggca cactgtgttt 52440 tgatctggaa attggaggtg aatcctgaaa aatggaaggc aagcactatc agtgctataa 52500 aggattgtta aagtgtagtg ccagtggact gatcccagca ctgcgcagtg gtcattatca 52560 ttttggtaat ctgaagtaga agcatgcctc tagacatcct tccataattt tatataggta 52620 gtgtaaggat gactgtctat aaattcacct gaactacaga gagacttctg atataataaa 52680 agaaaaatac agagattgtt caggatggga atacaaacac taggaaactg gaaaagcaaa 52740 ttaaaacttc tgaattaata gaaaaagtta aataggaaag agaaaataaa gggaaacttg 52800 accaaggtag caaagaaaga aaatggaaaa gaggtaatta tgaggcagta taaaacaaac 52860 tctgtcgcaa aaaaaaaaaa aaaaaaaaaa aaaattccat ttgtagtttt aaaaaaatag 52920 taaagatata gaaaaattag tagaaaatac aaaaatctgt attcttcttt tatcagaatt 52980 aaataattat gcctgcccta ttttaccaaa ttattttatt aaaacatgct aaagaagttt 53040 ataagctttt cttctccctg ctcccattct ctataggatt tttgctgaga tatttcaaaa 53100 tttttatttt taggttatat ttaagctttc acttctaatc tctctcaaaa acctttcata 53160 tcacttgtca taaaaatttc ttctcatagt atcatcatcc agattcagtc acatatacat 53220 caagaataat tgtgcaatac tgtttcctag actcttcatc ttatcttttc aatgcatgat 53280 atttaaataa gccatacttt ttaatgtttt caatttttta atttatattt atttatttca 53340 aattttttat tatattttaa gttctgggat acacgtgcag aacgtgaagg tttgttacat 53400 aggtatacac gtgccatgat gttttgctgc acccatcaac ccatcatcta cattaggtat 53460 ttcttctaat gttatccctc ccttagcccc ccaccccccg acagatccct gtgtgtgaca 53520 ttcccctccc tgtgtccatg tgttctcatt gttcacctcc cacttatgag agaacatgca 53580 gtgtttggtt ttctgttctt gtgttagttt gctctgaatg atggtttcca gcttcatcca 53640 tgtccctgca aaggacatga actcatccct tttgatagct caatgttatt gcatggtgta 53700 tatgtgccac attttcttta tccagtctat cattgatggg catttgggtt ggctccaaat 53760 ctttactatt gtgaatagtg ctgcagtaaa catacgtgtg catgtgtctt tatggtacaa 53820 tgatttataa tcctttgggt acatacccag taacgggatt gctgggtcaa atggtatttc 53880 tggttctaga tccttgagga atcaccacac tgtcttctac aatggttgaa ctaatttaca 53940 ctgccaccaa cagcgtaaaa gctttcctat ttctccacat cttttccggc atctattgtt 54000 tcctgagttt ttaatggtca tcattctaac tggcatgaaa tggcatctca ttgtggtatt 54060 gatttgcatt tcgaccagtg atgatgagct tttttttcat gtttattgcc cacataaatg 54120 tcttcttttg agaagtgtct gttcatatcc tttgcccact ttttgatggg gttgtttttt 54180 gttttttttt ttgtaaattt gtttaaattc tttgtagctt cttgatagta gtcctttgtt 54240 agatggataa attgcagaaa ttttctccca ttctgtaggt tgcctgttca ctctgatgat 54300 agtttctttt gctgtgcaga aggtctttag tttaattaga tcccatttgt caattttgtc 54360 ttttgttgcc attgcttttg gtgttttagt catgaagtct ttgcccatgc ctatgtcctg 54420 aatgatattg cccaggtttt cctctagggt ttttatggtt ttcagtctta catttaagtc 54480 tttgatctat cttgagttaa tttttgtata aggtgtaagg aaggggtcca ctttcaattt 54540 tctgtatatg gctagccagt ttccccaaca ccatttatta aatagggaat cctttcccca 54600 ttgctttttt ggtcagattt gtcaaagatc agatggtaga tgtgtggcat tatttctgag 54660 gcctctgttc tgcttcattg gtctatatat ctgttttggt accagtacca tgctgttttg 54720 gctactgtag ccttgtagta tagtttgaag tcagctagcc tgatgcctcc agctttgttc 54780 tttttgctta ggattgtctt ggttatatgg gctctttttt ggttccatag gaaatttaaa 54840 gtagtttttt ctaattctat gaagaaagtc aatgttagct tgttggggat agcattgaat 54900 ctataaatta ctttggtcag tatggccatt ttcatgatat tgattcttct gatccatgag 54960 catagagtgt ttttccattt gtgtcctctc tgatttcctt gagcgttgat ttgtagttct 55020 ccttgaaggg gtccttcaca tttctcgtga cttgtattcc tagctatttt actccttttg 55080 tagcaattgt gaatgggagt tcagtcatgt ttgggctctc tgtttgtcta ttattggtgt 55140 ataggaatgc ctgtgatttt tgcacattta ttttgtatcc tgagacttca ctgaagttgc 55200 ttatcagctt aaggagattt tgggctgaga tgatggagtt ttctaaatat acaatcatgt 55260 cctctacaaa aagacacagt ttgacttcct ctcttcctat ttgaatacgc tttatttctt 55320 tctcttgcct gattgccctg gccagaactt ccactactat gtttaatagc agtggtggga 55380 aaggacatcc ctgtcttgtg ctggttttca aagggaatgc tttcagcttt tgcctattca 55440 ttatgatatt cgctgtgggt ttgccataaa tagctcttat tattttgaga tacggtccat 55500 caatacctag cttattgaga gtttttagca tgaaggggtg ttgaattttg ttaaaggcct 55560 tttctgcatc tattgagata atcatgtggt ttttgtcatt ggttctgttt atgtgatgga 55620 ttacgtttat taatgtgcgt atgttgatcc aggctttcat ctcagggatg aaactgacct 55680 gatcatggtg gataagcttt ttgatgtgct gctggatttg gattgccagt attttattga 55740 ggatttttgc atcaatattc atcagggata ttggcctgaa attttctttt ttgttgtttc 55800 tctgccgggt tttggtgtca ggatgatacc aaataaaatg agttagggag gattccctct 55860 ttttctattg tttggaatag ttttagaaag aatggtgcca gctcctcctt gtatctctgg 55920 tagaattcag ctatgaatct gtctggtcct ggggtttttt tggttggtag gctattaatt 55980 actgcctcaa tttcagaact tgttattggt ctattcagga attcaacttc ttcctggttt 56040 catcttggga gggtgtatgt gtccaggaat gtatgcattt cttctagatt ttctagttta 56100 ttttcataga ggtgtttata gtattctctg atggtagttt gtatttctgt gggatcactg 56160 gtgatatccc ctttatcatt ttttattgtg tctacttgat tcttctctcc cttctttatt 56220 agtctggcta gtggtctatc tactttgtta atcttttcaa aaaaccagct cctggattca 56280 ttgatttttt ttttttaagg gtttttcatg tctctatctc cttcggttct gctctggtct 56340 tagttatttc ttgtcctctg ctagcttttg aatttgtttg ctcttgcttc gctagttctt 56400 ttaattgtga tattagcgtg tcaattttag atcattccca ctttctcctg tggacattta 56460 gtgctataaa tttccctcta aacaaacact gctttagctg tgtcccagag attctggtat 56520 gttgtgtctt tgttctcatt ggtttcaaat aacttattta tttctgcctt aatttcatta 56580 tttatgcagt aatcattcag gagcaggttg ttcagtttcc atgtagttgt gcagttttga 56640 gtgagtttct taatcctgag ttctaatttg attgcactgt ggtctgagaa actgtttgtt 56700 attatttcca ttctttcaca tttgctgagg agtgttttac ttccaattat gtggtcaatt 56760 ttagaataag tgcaatgtgg tgctgagaag aaggtatatt ctgttgattt ggggtagaga 56820 gttctgtaga tgtctcttag gtccacttgg tccagagcct gagttcaagt cctggatatc 56880 cttattaatt ttctgtcttg ttgatctgtc taatattgac agtagggtgt taaaagtctc 56940 ccccattaat gtgtgggtgt ctaagtctct tcataggcct ctaagaactt gctttatgaa 57000 tctgaatgct cctgtattga gtacgtattt ttaggatagt cagctcttct tgttgcattg 57060 atccctttac cattatgtaa tggccttctt tgtcttttga tctttgttgg tttaaagtct 57120 gttttatcag agactaggat tgcaacccct gcctttttta gctttccatt tgcttggtaa 57180 atattcctcc atccctttat tttgagcata ttgtgtctct gcctgtgaga tgggtctcct 57240 gaatatagca caccaatggg tcttgactct ttatccagtt tgctggtctg tgtcttttaa 57300 ttggggcatt tagcccattt acatttaagg gtaatagtat tatgtgtgaa tttgatcctg 57360 tcattatgat gctagctggt tattttgccc attagttgat gcagtttctt cctagcatcg 57420 atggtcttta caatttggca tgtttttgca gtggctggta ccggttgttc ctttccatgt 57480 ttagtgcttc cttcaggagc tcttgtaagg caggcctcgt ggtgacaaaa tctctcagct 57540 tttgcttgtc tgtaaaggag tttatttctc ctttgcttat gaagcttagt ttggctggat 57600 atgaaattct gggttgaaaa ttcttttctt tagaaatgtt gaatattggc ctccactctc 57660 ttctggcatg tagggtttca gcagagagat ctgctgttag tctgatgggc ttccctttgt 57720 gggtaacccg actttctctt tggctgccct taacgttttt tccttcattt caatcttggt 57780 gaatctgaca gttatgtgtc ttggggttgc tcttctcgag gagtatcttt gtggtgttct 57840 ctgtatttcc tgaatttgaa tgttggcctg tcttgctagg ttggggaagt tctcctggat 57900 actatcccga agagtgtttt ccaacttggt tccattctcc ccgtcacttt caggtacacc 57960 aatcaaacgt aagtttggtc ttttcacata gtcccatatt tcttggaggc tttgtttgtt 58020 ccttttcatt ctttattctc taatcttgta ttcatgcttt atttcattca gttgatcttc 58080 aatctctgat atcctttctt ctgcttgata aatttggctg ctgatatttg tgtatgcttc 58140 acacagttct tgtgctgtgt ttttcagctc catcaggtca tttatgttct tctctaaaat 58200 ggttattcta ggtagcaatt cctctaacct tttttcaagg ttcttagctt ccttgcattg 58260 ggttagaaca tgctccatta actcagagga gtttgtcatt acccgcctcc tgaagcctac 58320 ttctgtcaat ttgtcaaact cattctccgt ccagttttat tcccttgctg gtgaggagtt 58380 gtgatctttt ggaggagaag aggcattctg atttttggaa ttttcagcct ttttgcactg 58440 gtttttcctc atcttcatgg atttatctac ctttggtctt tgatgttggt gaccttcaga 58500 tggggttttt gtgtggacat cctttttgtt gatgttgatg ctattccttt ctgtttgtta 58560 gttttccttc taacagtcag gcccctctgc tgcaggtctg ctggagtttg ctggaggtcc 58620 actccagacc ctgtttgcct gggtatcact agcggactct gcagtacagc aaagattgct 58680 gcctgttcct tcctctggaa tcttcatccc agcagggcac ccaccagatt gccagccaga 58740 gctctcctgt gtgaggtgtc tgtcgacccc tgctgggagg tatctcccag tcaggaggca 58800 caggggtcag ggacccactt gaggaggcag tttgtccttt agcagagctt gagcactgcg 58860 ctgggagatc cgctggtctc ttcagagcca gcaggcagga acgtttaagt ctgctgaagc 58920 tgcgcccaca gccacccctt cccccaggtt ctctgtccca gggagatggg agttttatgt 58980 ataagcccct gactggggct gctgcctttc tttcagagat gccctgccca gagaggagtc 59040 tagagaggca gtctggctac agcagctttg ccatgctgca gtgggctctg cccagtttga 59100 agttccaggt ggttttgctt acactgtgag gggaaaactg cctactcaag cctcagtaat 59160 ggtggacgcc cctcccacca ccatgctcca gcgtcccagg tcgacttcac actgctgtgc 59220 tggcagcgag aatttcaagc cagtgaatct caacttactg ggctctgtgg ggatggtatc 59280 tgctgagcta gatcacttgg ctccctggct tcagcccccc ttccagggga gtgaatggtt 59340 ctgtctcact ggcattccag gtgccactgg ggtatgaaaa aaaactcctg cagctagttc 59400 attgtctgcc caaatggctg cccagttttg tgcttgaaac ccagggccct ggtgatttag 59460 gcacccaagg gaatctcctg gtctgtgggt tgcaaagacc atggggaaag catagtatct 59520 gggccggaac acattgttcc ttacagcaca gtcactcacg gcttcccttg gctaagggag 59580 ggagtttccc cactctttgt acttcccggg taaggcaatg ccccaccctg cttcagctcg 59640 ctctccatgg gctgtagcta ctgtctagcc agtcccaatg agatgaggtg ggtacctcag 59700 ttgaaaatgc agaaatcacc caccttctgt gttgatcgcg ttgggagcta cagaccggag 59760 ctattcctat tcagccatct tgccagccag caaccactct actttttttt tttttaattt 59820 aagttctggg atacatgtac agaatatgca ggtttgttac atagatatac atgtgccatg 59880 gtggtttgct gcacccaaca atccatcatc tacattagat gtttctacta gtgctatccc 59940 tcccctagct ccccacccct caacaggccc tggtgtgtga tgttcctctc cctgtgtcca 60000 tgtgttctca ttgttcacct cccacttatg agagaatatg cagtgtttgg ttttctgttc 60060 ttgtgttaat ttactaagaa tggtttccag cttcatccat gtccctgcaa aagacatgaa 60120 ctcatccctt ttgatggctg catagtattc catggtgtat atgtgccaca ttttctttat 60180 ccagtctctc actgatgagg atttgggttg gctccaagtc tttgctattg tgaatactgc 60240 tgcagtaaac atacatgtgc atgtgtctta tggtacaatg atttataatc ctttgggtat 60300 atacccagta atgggattac tgggtcaaat ggcatttctg gttctagatc tttgaggaat 60360 catcacactg tcttctacag tggttgaact aatttacact cccaccaaca gtgtaaaagc 60420 tttcctattt ctccacatcc tctccagcat ctgttgtttc ctgacttttt aatgatcgcc 60480 attctaactg gtgtgagatg gtatctcatt gtggttttga tttgcatttc tctcatgacc 60540 agtgaagatg agcttttttc catgtttttt ggccacataa atgtcttctt ttgagaagtg 60600 tctattcata cccttcaccc actttttgat gttttttttt tcttgtaaat ttgtttaagt 60660 tccctgtaga ctctggatat tagccctttg tcagatggat aaattgcaga gattttctcc 60720 cattctatag gttgcctgtt cactccgatg atagtttctt ttgctgtgca gaaggtcttt 60780 agtttaatta gatcccattt gtcaattttg tcttttgttg ccattgcttt tggtgtttta 60840 gtcatgaagt ctttgcccat gcctatgtcc tgaatggtat tgcctaggtt ttcttccaag 60900 gtttttatgg ttttaggtct tatgtttaag tctttagtcc atcttgagtt aatttttgta 60960 ttaggtgtag ggaaggggtc cactttcagt tttctgcata tggctagcca gtttccccaa 61020 caccatttat taaataggga atcctttccc cattgttttt tttggtcagg tttgtcaaag 61080 atcagatggt tgtagatttg tgacgttact tctgaggcct ctgttctgtt ctattgttct 61140 atatatctgt ttgggtacca gtaccatgct gttttagtta ctgtagagtt gaagtatagt 61200 ttgaagtcag gtagcatgat acctccagct ttgttctttt gacttaggat gtcttggcta 61260 tacaggctct tttttggttc catatgaaat ttaaagtaat ttttttctaa ttttgtgaag 61320 aaagtcaatg ttagcttgat ggagctatta ttggatctat aaattacttt ggtcactatg 61380 gccattttca aaatatacat tcttcctatc tatgaatatg gaatgttttt ccatttgttt 61440 gtgccctgat ttccttgagc agtgttttgt agttctcctt gaagaggccc ttcacatccc 61500 ttgtaagttg tattcctggg catttaattc tctttgtagc aattgtgaat gggagttcac 61560 tcatgatttg gctctctgtt tgtctattat tggtgtatag gaatgactgt gatttttgca 61620 cattgatttt gtatcctaag aataagccat actttttaat atattttatt cataaaaaaa 61680 tgcacataaa gcaaatgaca tatagaaaca atatgattaa catccacctc tggactcagt 61740 tggcttaaga aaatatgaat attatcacta cacttgaaat tgattgcata tttcttcctt 61800 atcacattcc tctttatctt tctccagaaa cctctatatc tttaatttgg agttggtcat 61860 tttcttatat tattctgtag gattttttaa tatctatgta tttctcacta atagattttt 61920 tagttttgag atgttttttt ctactccata gaaatgctat gtatatttat gcaatttgat 61980 cttactgcct aatattatat ttgtgttttt tattttgatt tgtattattc tggtttgttt 62040 tttcctacac tgtatgattt cattgtatga gtatctccca tttatttatt ctcatgtatt 62100 gacatgtact tccacttgat atttgctgtg ctttacctac agttcaattg tgaacattat 62160 tttacatgta ttctggtggt tcatgtgtga gagtttctgt aggtaacctg cacacctggg 62220 catgaaattt ctgactgtaa tacatgctca tgctgggctt tactagataa tgctagattg 62280 tttttaaagt agtagtagtt tacactccat ggcagctctt cattgttttg tgtgttcctg 62340 tattctgtgt ccttgccgtt ttaggtagat gatgtctcat gtggtcttac tttgcatttc 62400 tctggttaat aatgaggtca gtcatattgt tgtattttgt catctatata tttcttcctc 62460 tgtgaaatat ctgttcatgc cttttctttc tgatttatag gaattctttg tgtattttgg 62520 gtatatatgt tataaatatc tataggatag ataaaaacaa gctatctgat aaggcgatat 62580 gcagaaatat aaattaggtg aggaagcaag ccatgcaaat atccaaggaa aaaatcattt 62640 catgcagagg aacttgtcag tgtaagaccc caaggcagaa acacacttca agggcaagat 62700 gggcatcaat atgggtggag catgagggag aatagaagag agaccatcta tgcaagggta 62760 agttgggcct tgtagagctt gatatgaatt ttaacttcat tctaagtagg atcaggagtc 62820 actgaaaata tcaagcaaga atgacatagt ctgattaagt ctttaaagtt gtatagaaaa 62880 tagtctacaa aaagtgaaag ctgggaaacc agccagcata tgttacaata gtccaactgt 62940 gattgtgggg acataaatta tggtgcaagc agtggaaaca ggtatttgga tttgggttat 63000 attttcatgg ttatccacta ggatttgctg atgtattaga tatgaggtga gaaaataagt 63060 gaggtatcag taatgactct agagtttggg gctagagcag caggtgtgta aatgaagtta 63120 caaacctaga agatgcatgt ttggaccaat gcatcaaagt tctgctctaa acatattaat 63180 ttgaggggtc tgttagaaat acaagtggag atgctgagaa tacagttgca tagttgagtc 63240 tagagtttct tggaaaagct gggtctagga ctgtaaattt ggtaatcatt aatgtctaga 63300 tgatattaga gacatagaac tagatgcaga cacctagaaa gagcatagat atctaagaga 63360 agtctgagga ctgagcttgg tgtactcaaa gatttagaag ttgagaagat gaaaaagagc 63420 tagacagaga gactgaaaga gtgactacaa gaatgaaata tgtggaagcc aaatgaataa 63480 gagcgttaaa agaagaagga aacgatcaat gtgttaagta cttacccggg ttaagaccag 63540 gactgagaat tggccattgt tttttgacat ttagaaatct ttgaaggcct tgccaagaat 63600 aatttggttg ggataaggag aggaatgacc tgactggaga aggttcaagg gagaattgga 63660 ggagaggaaa tgaagagagt gatatttctc cacggaatac attatgtgtg agagggacta 63720 ttagaggatg cagacaagag ggcaaatgat ttcagagaaa agtccctgag tgggtgagaa 63780 ggactaagat ttggtattga aatgtcagga gtatgaattg taacaggagg gaaggcagag 63840 tacaggggta gccatactgt agattggcca caaagaaaat attctccaaa tggagcttct 63900 ttccccctaa gcctccccca aatcattacc tctgcttctg attcaatccc tcaaaacctc 63960 atccatcagc ttgaatgaga gacaactatt cagccaccat gtccatccgc tttcttatcc 64020 ccccttgctc acccccagcg tctcagccca aatcttccct ccttccagcc aacaaagctc 64080 ccatgcctgt catctcctga tgtcaatgca gcgggcaggg gagtggggga ccactgaccc 64140 cacatgccta ttctctgctt agattaccca aaataccagg tgtcgttatt tttcagaggt 64200 gcaaaaatat aatacaaagt cattctaaac tgtcactgaa ttcagagtac tttttgccat 64260 tgtctagtca ctgaattcag agtacttttt gccattgtct tattttctca atcaacaaat 64320 gctaagagta caattatatg aagttctaca gatttatttg ttacttgcat tacttagtaa 64380 tggttcttat gcataaaggt taggaataac ttctttgaac aaatatccta tataaaaaaa 64440 caacaaagtg gtttttaaaa atcacattgc tgttcacatt ccctttttag tactctcttc 64500 tcatattctt ttttaagcta tgttaggtca aaacaactac tttaataact tagcaaatat 64560 ctatcaggag tccactgtgt agttagcagt ttataaagct tcagatctac attagtgagt 64620 gaaatggacc tagcccctat ccttacaggg cagcaagcct ggtggaggaa gctatgttct 64680 ctgaaggcaa caaaaacagt catgcgtcat ttaagagtag ggatacattt tgagaaatgt 64740 gtcatgaagt agtcaatttc atcattgaac atcatagaat gcatttacgc agacttagat 64800 ggtatagcct actacacacc taggctgttt ggtgtagccc attgctcctg ggctacaaac 64860 cgatacagca tgttactctg ttgaattttg taggcaactg taatgcaatg gtaagtattt 64920 gtgtacctaa acatatctaa agatagagac agtacagtaa aaaatactat cttatgaaaa 64980 cactgttgta tatgtggtct gtcattgact gaaacaatat tatgtagtgc aaaccataca 65040 gcaacagata ataaagacaa gatattcaga gtgggagatg gaaaagacaa taatgtattc 65100 cttcattcat ttagcagaca tgtattgcca aaagacactg gaaaaaataa ataaaggtga 65160 caaacaggtg gtgcctttgt tgaagattat agtcatggac agggaaactt tagccctctt 65220 cttaacatta tcttccctta gacacaaaca cacacacaca caattcctgg catatgctat 65280 atactccata actgttcctt cccttccctt tgtgactcat tccttccccc agaaagctca 65340 tttgatctct gcaaataatt ttgaatcccc tttgtttttt aaccatggca attgttttgt 65400 tttcccatct cctacaagtt ttagctcaac tcgttggggc tgtttttttt tttttttttt 65460 tttttttttt tttttttttt ttgaggtgga gtttctccct tgtcacctgg gctggagtgc 65520 aatggcatga tcttgactca ctgcaacctc tgcctcctgg gttcaagtga ttttcctgcc 65580 tcagcctctg gagtaactgg gattacaggt gcccaccacc acacccagct aatttttgta 65640 tttttagtag agacggggtt tcaccatgtt ggccaggctg gtctcgaact tctgacctca 65700 ggtaatctgc ccacctcggc ctctcaaagt gctagaacta caggcatgag ctcaccatgc 65760 ctggccattc cttggggctt tatagaacaa gtataatcct ttttctttat gacaataccc 65820 tgagttcaga aatctcagag acaaatgttt acagagacca ggtaaataag tagctagagt 65880 gacaagactt aagaaaagag accattggcg taccaccagt ttgtggaggg aactggaaaa 65940 actggaatac acatgcccca tccaaaagca accattgcaa ctaaacttta acagattgtt 66000 gccacctaag taattcacgg atggtctcat aattctggtc agcattgtct gagccaaaca 66060 aaatgtatct atgggcatga tcagatacta gagccagcag attgcaacct ctgcttagat 66120 aattgcaggt atcagccttc ccttggctaa acagctactt catactgata agtagccctt 66180 gcctggcaca aagcaggtgg ggctgaatcc agcctgatat cacatcacca caactttctc 66240 taattctcct caaggcgtct gtgaactacc agcagcccaa ataagcatcc ttttctcctt 66300 caatcatttc tcataaagca gattctactc atctcaccat ccatcttttt cccctaacat 66360 atcagaattc atcaaagtag aaactccaga aatcatccag aaatacaaat gtgatctgac 66420 cagcacgggt ataaactttc tctttcctta atttagaaaa aggattatta attttgagtc 66480 ttagatctca taaaagtaga gctggattga ttttaaggga tcaatgaacc tactgaaatt 66540 gtataaaaag attttgtggg gctggcaaac aaatgctttt cttttaacat attcttaaaa 66600 gaacaaaacc tcaaagaaga tttaacaacc actatagact aagtttcaat taatgtagtc 66660 tgtagcatta gattttggaa ggctgttcta tcagaatatg gaccaatact gagcgtagtc 66720 gctactaaaa ctctttgtct ttttattcat atcttctcct aaaaagctcc atgacccctg 66780 cagaatactg gtataattac ccacacctca cccacccagt gattgttgat gtaactaatt 66840 atttaatcca agtctgaggc ctaaattttt tttaatcaag agttagttac atgttttaat 66900 ttattttttg taattttaat atctaacact tttattctat ttcattcgat tgatgctaaa 66960 atgcataatg ctatgattct aagacttagg aaaaataata ctatactggg atatggagtt 67020 tatatatatt tacatgaatt tctttttttt cttctctgta ggtcgaaaat ttaaaaagtt 67080 caataaagtc agagttgtga gagcactgga tgctgttgct ctcccacagc cagtgggcgt 67140 tccaaatgaa agccaagccc taagccagag attcactttt tcaccaggtc aagacataca 67200 gttgattcca ccactgatca acctgttaat gagcattgaa ccagatgtga tctatgcagg 67260 acatgacaac acaaaacctg acacctccag ttctttgctg acaagtctta atcaactagg 67320 cgagaggcaa cttctttcag tagtcaagtg gtctaaatca ttgccaggta ataataattt 67380 ttatatacag catgtaataa aaaatatact atgtttgttg tattagtaca ttgttaaata 67440 aaacactcta tgcagtaaat taactacaat ataaaacagt atgtgatagt atgtgtttga 67500 gcagttcaaa atatttgtct aggaccctaa tatgtgttac agaaatataa aaaattagag 67560 aacacactga gtcttactga tgagtcagaa attgcataca ttaaacttat gctaataaga 67620 aatagcttag gtttatgtgt tcaaccacag atattagagt agctcagaaa tgataaatgg 67680 accatttttt ataaatgccc tatttattct aacccttaat tcaaccaacc tcatttggtt 67740 gaaaataaaa ctgaatgtat aaagtcatat aacaaatcaa agtagtattc agagtacagt 67800 aaggtgtcaa aagtaaatta agaaaacaaa attccctaac gtaaattact ttaaaaaata 67860 atagcacaca gagaaaagaa tctctagtca ctattcctct aatttatacc caaagagagt 67920 atatcgccca gcaacagaga gaacttggat tatatcatca cagaagaatt tcagtcactt 67980 tacttatcag ctatttttgc tccctcagca cagctctgta ggagccaagt acttgaaaga 68040 ataacaagga tgaaattctc acttggttaa atcttagact gcagagccag gagaatacat 68100 gtatagcaat tgtttgtgaa ggcattactc ctataattag aaatagaact ttttaaaaaa 68160 ctacattctc ttcacatcag taataccctt atactgctat agacaaatta tttttctaat 68220 atctgatcaa gaattaaaca tattatgtcc ttttagtccc caagtaaatt caatggcact 68280 tttagaaaaa gaatttcttc ccaattctcc ttaatataat tctcagttat tattctacct 68340 aaaaggtttg aaatacaata gatgaattgt tactgaaaat tcaattacat gaaacagaaa 68400 gaaaaataga tacctgtttg ttgaatcttt caaaaaatac atattaagaa aagttaatag 68460 aattcagtat tctagtggag atgaagtaat tttatagaaa aatcaaaatg cccagattct 68520 tactggcttc agtctcacca aaaggcctta aacagacaat aattttttga acattctaaa 68580 tgtttaagat ttatcacata gtggttatca aacttgacag agctttaata aataaaaata 68640 acccaatagc attgatgaac tttttctgtt taaaaatgag tgtttaaaca tgtgtttaat 68700 ttttctccat tgttctatta tttggaaaat atgatttctt tggtttttgt tgatatagtt 68760 tatattcctt ttgtttgcaa ttttggaggt tctttactat ttaaaaagat tctagcatca 68820 gctggtaacg ttagaaaata accagttata cttgaaaaga tgacttattc actttggata 68880 aaagctaagt gaccttttta accggaaacc gttatctatc aggacttttt gacccccaca 68940 caaccacaaa aaggaatgag agaaaactct tacattgaat ttggtggcaa cttttttaat 69000 gctgcaaaat gtaatttttg tgtagtaaat cagaataatg gcaacactag tattacccat 69060 actgtttcca ttaattgcaa acagggaaac attgagatta tcaaagtctc ttgtggcctg 69120 tggactttta aagccatatt ttcatggtct gcattttggg aatagcacta ttgaatgaat 69180 tatgaaaatg gtcagtgagc tagagtgact atttgacctc tgagaaagga cttggatgga 69240 cacatgtata gaaagtactt tagtattatt agctacttaa aactgatttc aattgacttg 69300 aaataagctt acagttttta ttcacttatt cactcaataa atgtctatta tgtacctgct 69360 gtgttcaata tctaataatt atacagtggt ttagaaagaa aagccacaag gctgggctca 69420 tggtgtggat gcattttggt acagcacatg ctagcatctg gtcatcagtt ttgcatgtaa 69480 aagaaattta tttcgttcat tcactcatgt gtaataaaac taataagtct gtggaagaca 69540 gacttttgtt aagtgatcct cactaattag ccagaatgtc aactaacttg cttatttaca 69600 gtgagacaca cacacacaca cgcacacaca cacaatataa atatacataa tacttatgtt 69660 gctaggagaa tatattaaca gatcagatca ttctttcaca ataggaaact gtatttggaa 69720 agtcacatta agatctgatg cagaatgacc atcataacaa aggtgacagc gtttatgaag 69780 acacctaggc atatatttgc aggtgcccta agaattctta ttgcctggta gggctttctc 69840 atttcttctt tgtctacact cttccattat cttggatctg ttataataat ggtagtttat 69900 atttcttcaa aaatatttca gatatatcct tttgtttctt ttcttttttt tctttttttt 69960 tttttttttt tgagagaggg tctcactcta ttgcccaggc tggaccacag tggcatgatc 70020 ttggctcatt gcagcctgga cctcttggtc tcaagttatc ctaccacctc agcctcctga 70080 tgagctggta gctgggacta caggcatgcg ccaccttgct tggctaaatt ttttttattt 70140 ttagttgaaa ttgggtttaa ctgttttacc caggctggtc ttgaactcct ggactcaagt 70200 gatctgcctg cctcagcctc ccaaagtgct ggaattacaa acatgactca ccgcacccca 70260 ccccttttac tttaatagag acttgcaaga tgctctagcc ttatgtcagc acgtttataa 70320 accatctctt aggatacatt gcaaatgagt gattttgcat gggcttttat ctgtagcctt 70380 agtgggcaat attttcttat ttgaaatgta gagcaacacg atatttgaat ttttttaatg 70440 aacaagacat attccctcta ttaaggaact ctcaatccag tagcagagag aagataccca 70500 aacaactata gtgttatgtg aaagtgatat aagatacaga tataatgaaa gtggggacag 70560 aagaacattt ttggcttcaa ggactaaaga agccttcaga gacaaaaatg tcagttaaac 70620 tgcatcttac aggagaaaca agactttgat aaatcatgat aagagaaagc attctagata 70680 gaaggaacag tatggctgaa ataagaaaag catgagatag gtttgctgaa taatgcatta 70740 aaccagcata ttaatggttc tcaacctttt caagtgtggg gggtccctta gtaacatcaa 70800 aaattttata gtctccccat agggtagtaa ttaaagcttt tagtagctat ggattgagaa 70860 aacacatttg tacatacagt ccctcggaca ctgctacagt aagtactggc cttcgggaga 70920 tttaatcacc caaatggtaa gaaaccattg aaaagaaatt tattttgaac tgaataatgg 70980 aaagtaaact tagaggtgta agttataggc tgatcaagga gggtcttaaa aattactgta 71040 gatgttagag tttaaattaa aagtactaat tcacaattcg gtgtcacctt taaattgtcc 71100 tcatcctgta catcaatacg ttgcagtgaa attgatgtga cagagggggt atgagggaaa 71160 cacttccgtt gcataaattt tgtcttgata tttcagttaa cttagtgaaa actccattta 71220 ggtcaatgct ttgaattttt aaaccttgca ttttttagga catactttat tctttgtgtc 71280 atttcaccag actacttgca attatcagta tcagaatatt agaacagtat tttgcctcat 71340 ttttctgctt ttgaagcgtg ccaaaacaac ttttgagtca gtgagtcatt gcatcttgac 71400 agaggatttt ttctcttcct tcccgaatca gaagactaaa ttaaattatt gctctccaga 71460 aaaaaatgat gagagagaaa tgagagtagc atactgagag taaaggcctt aataaatgtg 71520 ttgataaaga ctaaaccatg gtttttctca caccctattc cactgaaatt atgattgcca 71580 gtggcaacaa aaatttttac ttgtcaaatt agatgaatat ttccagccct taatgtgttt 71640 cacctacctc tgggactatt ggcaaagtaa ctatttcttg ctaagactct tgcttaggta 71700 tttgtcactt cccttccagg tagatatgtt tctctcccta tgtacatttc ccgactcctg 71760 tgtggatttg ttcttccacc cacactataa atttgtttgt gttctgggtt ctatcttttc 71820 cagtcatttc tcactctaca cagtctgcat aagctcattc atagtcttgt cttcaactac 71880 cactatattt cgatgactcc taaatacata atattagcca gaccctctac aaaactactg 71940 aactttcaac tgcttattac acagctccac ccagatgact tctaagtcag catgaccccc 72000 taaacctaag gccttttcca gtatcttgaa atggcacctc agcaagaatt tttggtatca 72060 tccttggctc agtttctcct tcacccctta cccaataaat tgtcaaattc tatcaactcc 72120 acttattaaa tatatgattc attatacatg ctacttctgt tgctacttcc ctgacttagt 72180 ttggtgctgg tcttttttta cttggattca tgttgcagcc tcctgactga ttctattacc 72240 atcaatttca cgtggtgccc aaatgatcct ctattctacc cccagaatca tttttctaaa 72300 acaattttaa tcaatcatat tttccatttc tttttccaaa tagcatccaa actccatggc 72360 ctcaatatcc aagggctttc tgtgatctgg ctcctgaatt tgcagtgtta atctctcatc 72420 attcctcttc acacgactct aagatctagt tccctaaatg ctgtttgctc aaattctagc 72480 cttcttctct tgcccatatt atttttccta cctggcagcc catctctttt cccacgtctt 72540 cctttgactg acctgtaact atctttcagg attcgcatta cttgtcactt gctctgggaa 72600 gatttctctg agccttccta gactgggtga ggaaaccctg taccctccct tgtctccttt 72660 gtgcttgtca accataacac tatgactctg agttacaatt gtgttctact tttctatctg 72720 tctcacaagc ctatagtgtt aaaggcagaa acagcatctt tctaaccttg atcctaattg 72780 caagtataat gcttcctccc aaatagatgg ttaaagaata tttatagaaa tttaaattac 72840 agaattgatt tgcaaagaac tgtactacct tagattatta gacattaact tcttattcag 72900 cataaatcat caaacatacg ttggtgatta tacttaatca tattgaagct tctttgtgct 72960 tctcaacatt gatacttgtg tatgaaagtt gttgctttgt gtcagaaggt gctgagatat 73020 ttaatgctct gccattgttg tagattatct gtttcaatta catgacattt tttacagaac 73080 caaaacctac atattttatc aaaactctgg gagtaagatt ggggaactta gtagtactgt 73140 catacaggcc atcttttttt tttttttttt tcttgagata ggctctcact ctgtcaccca 73200 ggctggagtg cagtggcatg attgtagctc acggcagctt agaactcctg ggctcaagcc 73260 atcgtcctgc ttcagcctcc ccagtaactt cagcctcccc agcactacag gtgtgcacca 73320 ccacacctgg acaagctttt taaattggtt tgtagagact gggtcttggc tggtctcaaa 73380 ttcctggcct caagtgactc tcctgcctcg cctcccaaag cactgggact acgggcatga 73440 gccaccacac ccagccccag gccatctctt cactatatac tttctgttaa ttctttaagc 73500 taaatttgga cagctgtaac agggcttttg tcagaaatta tcaggtcaag tcacttaaaa 73560 ggaaaagtgt tttttttttt aacatttgtt aaacacaatt agtgataatc catcacttct 73620 ccatgtgaag gtttttcatt ttggtcatct caaatgtact aagtttacct gatcattatc 73680 atcactggga atttaatctc tgtgttccca tctttgtttc agcataaagt aatgaatgat 73740 attacagtta ataaggaaaa aaatagcatt tctatactag gcatgttcaa gtcaatttaa 73800 cattgtacat ttatcacaga taagcactgt aaaaggtgga gtaaataaag gttattttct 73860 gagttctatt ccattctgtc aaacaaacag aaatatagtt tctgctataa atattggtaa 73920 ttttataaat ttaaagttat tgtaataaga tttgccttta atgttgtcat aaaataaaag 73980 taagcctgga tttctagtcc cagaatgcaa aaacaaagaa aaaagcaaaa agacgaaaac 74040 aaaaactaag agtaggataa agtgaatgta aacatgtaaa tgtagacagg atctgttttt 74100 ggataaagaa caatttaatt cttacataat tttgtaaaat gtttaaatgt ttaaaactac 74160 tcaaaggtta acataaaaca aatgcctgaa gtgaaaaatt acagcatagt taagtcaaat 74220 atttaaatat tataaaatac tttttttaga aagttataaa gttcaccaag aaagatatag 74280 agaataataa atgtatttgt ggagattatt gtgaacactt ggttaatgtt ttaatacatc 74340 tgattaatga aaacgaactt tctatgagcc tttttttttt tttttttttt tttttttttg 74400 agatggagtc tcgctgtgtc accaggctgg agtgcagtgg cacgttcttg gctcactgca 74460 acctcagcct cctgggttca aaccattctc ctgcctcatc ctcccaagta gctgatgaga 74520 ctacaggcat acaccaccac acctagctaa tttttgtatt tttagtagag acagggtttt 74580 accatgctgg ccaagatggt ctcaatctct tgaccttgtg atccacccac ctcggcctcc 74640 caaagtgctg ggattacagg cgtgagccac tgcgcccagc ccattttttt gtattatatg 74700 aaatatataa tttgttatat ataacaacac acataaaata tatattctgg aaaatatata 74760 ttccagaaca aacacataat ttatctttaa aattttttca ccattatttt ttatacttca 74820 gttctatctt atatgttcta taatatataa aatgcctcct aacacttttt tttatggcat 74880 tgtgttttag tattagagac ttcccattaa actgactggt ggatttcata ctagtattac 74940 tccaatgagc aattctgttg cattgataga ccaataataa atatctccat agtgttctac 75000 agtgtgcata attatcccat ttgtctcata gcaactcact gagttagtta tcattgtgaa 75060 ttgtgatttt tacttatgaa gaaatttaga ttcaatgata ataagtgatg tttccaaagt 75120 ggctgttagt aagtggtaga gccaggactg gaacttgggt cttctgtcct gaatgccttc 75180 caatttgttg agacagatca gcaataggga ggtttatatg tgcctccctt tagaaaggct 75240 tccttttcaa aaattaagac aattcattga tctcttgtca aatgattcta aattttatca 75300 caaaattacc catttttata gtctcttaaa tatttgactt acaagcagca tttatgtatt 75360 agatttattg tattgagctt tataattttc ttaaaaagtt attatgtaga cctatacttg 75420 aagacacgtt caagtcatat gcaaattaaa aaatgataaa acttgcatac caaaaatttt 75480 tttggctgct tagttagaga gtctttctgc acctgagtaa accaggattc ttggatgcca 75540 gtattaaaaa taaactatgt taattataaa acacacaagc aacaacaaaa aaccctgact 75600 tttaaaaaca aaagaaaaag aaaaaaaata atttactgag aggacaacag gggttcactg 75660 aatagaagct agaggacaag cttagaatat gggcagaagc caaggcacat ctgaagggct 75720 aaaaagcaag catcacaaat cctcccattg gttctcatta tctggacacc accaacatca 75780 agaataaata gtcatatctt tctctttgct ttagtatttt ctgattcaaa ttcctgttag 75840 tatatgttcc atgggccaaa cctatgcccc aagcccaaag aaggaggagg tttacccagc 75900 tttctccgtg ggaaagtagg aaggtaaggc atttgctctc gcaaaaacta caaccaatgg 75960 ggaactctgc ccacacagag aaaggaagga ggggaggaag gaaggaggga aggaaagaag 76020 gaaagcagga aggtattagg gagcagtgag ggaggaggag gaaaaagagg aaaggaagaa 76080 agagagggaa ggaagaaagg agggagggag gaagggagtg agagaagaaa ggagggagaa 76140 ggggaagaaa gaaaggaggg aaaaggggag ggaagaaagg aggaagaagg ggagggaaga 76200 aaggagggag aaggggaggg aagaaaggga gaaggggagg gaagaaaggg agaaggggag 76260 ggaagaaagg gagaagggga gggaagaaag gagggagaag gggagggagg aaaggtggga 76320 aggagaaaag gttagacatt gaagagcatc ccttctcagt aaaaggtaaa tgctcactat 76380 ggcatctcta attatgaata tgcatttgag tttttggtct tattgaggct aaggagttct 76440 aagaagagag tagcaatttt tctactattc acatgaagat gggacgatct gaagtgtatg 76500 aagagaagag ccttcttttt tgctacctac cttgacatga ttaacattgt caggaaataa 76560 ctttcttttg ctcttgatag ctaatgaatt aagttttgct ttggttttac ttttgaaaca 76620 gattatttgt aagaacatca gtggattttt agagaaaata gatgttttta aaacagcttt 76680 atagaaagtg gttgatgttc tcagcaaaac attgtctcca tacctttaat tttaaaaatt 76740 ctccattttc atcatttcag ttatactcag atacaaataa ctttcaaatt tatattaata 76800 tttttagccc atatatctcc ccatacttct aattcatatg tgcagctgat accaggatgt 76860 tgacccctga atcctatcca aaactatttt ctccctagca agtttcttct cctttatttt 76920 atctcagcaa tgctgttatc atcatctacc cttcaagaca aatactgaaa gatgtatttg 76980 attccttctt cttcccatct accggcaatc agttgacaaa tgtcttttat ttttcccttt 77040 aaatattttt ctgatgcact ctctcctctt aattccaatg tgttgacttg acttacttca 77100 ggcttttatc atctctcacc tgggctttga cagtatctcc caactgtcct ccctaactcc 77160 agacttattc ccttgaatcc acctaccaca tagctaagaa tgatccatct acaatgcaag 77220 gctatgtcac tcccattgct ccttaataga cttgcaagac cttggatgat ctgacccagc 77280 ctacatctct aaacacatct ttgcttctcc cacctttaag ttactcattg ttgaactggt 77340 gatcttccct tgagaattgg gaagacatgg agagacttgt gatgattaag tcaagattca 77400 tttgcccttt ccctgccatc cttctgccac caacaccatg gaacagccta aaagaggatg 77460 aaagaccaaa gcattcacac catacccccc aatacacaca cacacacaca cacacacaca 77520 cacacacaca cacacacaca cacagattct aaatctctta agccacacgg aagtttaaaa 77580 caagactgga ttaagtttca gagaacgaaa gtgacctgat gactgtggaa tctacccgac 77640 atagttaagg gttgttaaaa gaaaattaga tataacacaa ctgaaagcag tggttttaca 77700 aagtaagacc agtttatgtt taaatcccaa agaactgaga tttaacgaac tgattatatt 77760 ccattcaggc attatctctc tttttttcta gaaagttttt cctgacctcc ctcccgattg 77820 actgatacac tcccttttaa ccttctctac cccagcactc atgtgcttgt ttctcctgtt 77880 gagttgtgag ctgcttttga ggctaggatt gactcttact catcttcatt tttatacagt 77940 gcctggcacc caaatggttc ttattaaatg ggtgttgcat gaatgagtga tggatgatgg 78000 attcacttag caaaatccca gaaatgctaa atcacagtga ctaatttcaa gtgacttcca 78060 aagttgctga tattttaggc tgtattattt attaatgttt tctgattttc agtgtaaaat 78120 atgttgggat tgaaaacatt ttattaccct acgcatacct attttcagat ttatatgtat 78180 tttcaacatt tttgagataa ttcagtcatt tttatggtca ctgtattttt aaatttgttt 78240 tgtaggtttt cgaaacttac atattgatga ccagataact ctcattcagt attcttggat 78300 gagcttaatg gtgtttggtc taggatggag atcctacaaa cacgtcagtg ggcagatgct 78360 gtattttgca cctgatctaa tactaaatga gtaagtagta acttttgttg tttttgttat 78420 tttaagtgta catgtaggat aattttgaaa gttatatttc aatagatgat cacatattta 78480 gtgttcttga tatgatgaca ttactgtcat tttacggtaa ttttatattt ggagcttttt 78540 cctcttgttt caaaatatag ttatatcaaa tccaattttc ttacacatca agaatgtatc 78600 aagaaatata tttaaagata aacttaaatg tttcattaat caatttaaaa tttcatagct 78660 tggtactaat gacaataata aatgagaaat atttttgtta ctgacttaca ttcaaaaagt 78720 aaaaacatct ttcttcatac atgacaaatt agactgctaa tttatttaaa aaattgaaag 78780 ataaatattt caatttgctt tatatttaaa taattttaat taattttcta cctacaacct 78840 ttaactgaat aagaatcttt tcatggtaat atttatattg ttctagtggt aaatgctgac 78900 ttagaagtat ataactcaca cttatataag tctttacagt ttttagataa ttttcatgtt 78960 tcattacatt ttatccaaca attccttgag ataagtaagg aatcagcatt cttcattgta 79020 cagtttagaa aactgagtct caggagaatt atataatctg ccaaataatt tgaatgacag 79080 tgagattcct tatctctttt tagccaaaat ctagttttca ttattcttgt tagcttcatt 79140 tgactaatgt ggacattata aaacaacaca aggttgggaa aatgccttaa ttaaattttc 79200 tcagccttca gcacttgtgt ttaactttgt caattaatta atgcttgcca atgtcctctt 79260 ttactctttt ctgtatcatt gtatatttcc ctagaaaagt ttaaaatatg gtaagctttg 79320 tgaactgact tctcctattt aattgcacac aaaattatat aatttttcat agaaaataaa 79380 ttgtaagtaa ataaaacatt tctgagcatt cttcagcttc acattctcaa ctcctacacc 79440 tctgctgtgt tttcttggtt taggtattta aagtgtgata ataaaagtga gaaacaatgt 79500 taatcttggt cactgtgtat ttaatataat tttctagttt taagatttct taacctcaaa 79560 aattcagtat tgtgaaaagt ttagaagatg tttaaaattc caaatattgg gtttgtttat 79620 taaaagtagt agtcattttc aataatatgt aaggtaatta ccaaaaccat attccctgat 79680 aaattaccta aactggctga gacactattt tttttcctgt tgctttttct tttgtgtatt 79740 gtgtgtgatg caagacagcg gatgaaagaa tcatcattct attcattatg ccttaccatg 79800 tggcagatcc cacaggagtt tgtcaagctt caagttagcc aagaagagtt cctctgtatg 79860 aaagtattgt tacttcttaa tacaagtgag tgagttcaag taacttaatg caagatatct 79920 agtttcttaa ttcattagaa aagttgcaaa caatatgatt atatagttat gtatgaggta 79980 gacgtcttgg attataagta taaagaagaa atacccaata tattgttata gacattaata 80040 aaattactgg attttttcat cttttaccta ccatataata ctaaaatagc ctcatcaatt 80100 tctttttatt ttagataaaa tgatttaatt actctttcat attcacctaa ttcagtaata 80160 taaactacag tcatgtgcca cataataata ttttggtcaa caacagacca catatacaat 80220 ggttccctaa gatgatagtg gagctgaaaa attatcatcg cctagtcctg ttatagccac 80280 cataacatca tagcacaatg cactaatcac atgttcgtgg tgatactaat gtaaacaaac 80340 ctactgtgct gccagtcata taaagtatag cacatacaat tatgtacagt acatagtact 80400 tggtaaagat aataaaaaac tcttactggt ttatgtattt actgtagtat acttttctat 80460 tgagagtcac tacttctact ttttttaagt taaccgcaaa acagcctcag gcaggtctgt 80520 cagagggatt ccagaaaagg gcatacgtca ccctgggaga tgacaactcc gtgtgtgtta 80580 ctgccccctg aagaccttcc agtgagataa gatgttgggg tggatgacag tgatattgat 80640 gattctgact ctgtgtaggt ctaggctaat gtgtgttttg ggtattagtt tttttaaaaa 80700 aagtttaagc aaaaaagaaa atatttaaaa atagataaag tttatagaat aaagatatag 80760 agaaagaaag ccaggcacgg tggcatgtgc ctgtagtccc cactacttgg gaggctgagg 80820 tgagaggatc acttgagccc aggagtttta agtccagcct aagcaacatg tgagaacttg 80880 tctctaaaaa aagaaagaaa gagaactgta ccttgtgttt ttgtttttaa ctaaatgtta 80940 ttacaaaaga gtcaaaaagt taaaaaacta gagtttataa agtaaaaaag ttacggtaag 81000 gtaaagctaa tttattataa aagaaataaa aatattttta taaatgtagt gtagcctaag 81060 tgtacagtgt ttataaaggc tatcgtagtg tatagtaatg tcctaggcct tcacattctg 81120 tcaccactca ctcgctgact cacgcagagc aacttccagt cctgcaagtt ccattcatgt 81180 taagtcccct atgcaagtgt cccattcgta tcttttatat tggattttta ctatgcctct 81240 tctgtgttta gatacaaaac acttaccatt gtgttacagc tgcctacagt attcagtaat 81300 cacatgctgt acaggttcat agcctaggaa taaggcctag gtgtatagta gtctatacca 81360 tctaggtttg tgtgagtaca ctctgtgata atcacacagt gatgaaatca cataaggaca 81420 catttctcag aatatatccc catcattaag tgatacacga ctgcaattaa tattggataa 81480 gcacaaacca tactctcctc taccaccact ctcaccccat ccccaaagta tttactatat 81540 aaaatacata attttgttta gaattatttt tatcaagata aattgttaaa acatgtaact 81600 ataaagatta gcatcttaat ataatagtgg tttctttatc actgacacag tccagtgagt 81660 tagcaagaaa cacccaggct ccttctagcc agtggcttca ccccttccta gtggcttgca 81720 aacctctatt ggacatgact agaaatgagc actgagtgag agagattttg aaggctagat 81780 ctgaaactat catacatcac tttcacccac attccaaggc acttaagctg caggaaaagc 81840 tgggaaatac agtctagcta taggcccaga agaaagagga aattatgttt tataaagcat 81900 tgcattgatc accagaagga caggcaaata tccgttccac tttttcttat atccaaggaa 81960 catacttacc tctgcctcag tggatcccca agggtctggg tgggcaaagt tttaaaaaaa 82020 agaaaaagca gctgctttga cccttgcata ggcaggatca gccctcaaat gtacaaactg 82080 atgcttttgt gattcattgt gatttctatg acaaccacag ggagctaaga cctcaggccg 82140 agggtgagcc taaggtcttt cttggcaaca tctttacagg gacttctttc taaggtccca 82200 aatctcaaat ggctaggggg tgcagatcag gagggaaggg caggagagat aaaccctgaa 82260 atggaaggct tcctcataga atttatgcaa agacagggat gaatacataa ccatcttggg 82320 aaccctattt ggacaaggaa tttcaaggct aatttggtga cagagcatca attgaacaaa 82380 ggatatttac gtggaaccgg cccagagagt caactggaat acatttaata atacagtaat 82440 tagctcctgc ccttctttgc atgaaggaat gaatcctata gcacttatgt cacagatagt 82500 cctagatctt ccaaactggc ttatatcctt aacataattc tctatttatg gataggcaac 82560 attatgctgg ggaattaaac aacaatggaa ttaatcaaca gggaggcttt gctcattgtt 82620 gtcacttaga aacttaggcg atcactagag cagtggtaag aaaaggaagt gagattcata 82680 caacttccac tcacatacca tagagagtaa gatttttagc catttgccac ttcacaaaaa 82740 ggaagaaaaa gaaactattt aatgaatagt acttacgctt tccacaaatt ctaatcaaga 82800 ttctaatgag atttggggag aagagggtga acttgacaaa cctactgaaa atccatcttt 82860 aaaactagaa gaataaatag ataagcatta taaaaatatg cagggaatta gaaagctgct 82920 gttaattaat acagcatggg agaagagata tactgaactt ttgggcaaat cagacaagta 82980 tatattagaa tataaatagt atagtggaaa ccacaaatca atgagtctga aacaaataaa 83040 tgttaagaaa aatagtttta tatttggaaa aaaactgttt ggatatgtag ctcaaattat 83100 atccttgaat aacagataat tgagttaaat gtaaacactc aaataattaa aaaacaagct 83160 agaagaaata gaaataatct tcaatctcaa atacctttta taaaataaat gcaaggagaa 83220 gattggcaaa gtaaaacttt gataatttgg tagttatgaa cttaaacctc tcatttgcca 83280 ttaaatatga aaatataaat ctccaggaat agacagtagg ttgaacaaac atttttacat 83340 tgttctctcc aaaattacac taaaggccag atgtagtcgg tcacatttct aatcacactt 83400 tgggagggca agctgggcag atggcttgaa cccaggagtt tgagaccagc ctaggcaact 83460 tggaaaaccc cgtctctaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 83520 attagccagg cgtagtggca agtgcctgta gtcccaggta gccgggaaac tgaggtggga 83580 ggatcacctc aacctaggaa gtcgaggctg cagtgagcca tgattgtgcc actgtactcc 83640 tgcctgggtg attgagtgag accatgtctc aaaataaaaa taaataaata aataaataaa 83700 aataaataaa ttacactaaa atcttagtaa agggatttac tatgaaaaat taagaaaaaa 83760 tgattttaca ttcatgttta cagctgaggg aaatataggg attggttggt aaccttaaag 83820 agagctgtga aagtttgaaa tgactgctat ttgaagtagt ccagaaaatt agtacagcct 83880 ctatggaaca ccatatggag atttctcaaa aaactataaa tataactacc attctatcta 83940 gcaatcccac cactggatat ctacccaaag gaaaagaaat cattatgtca aaaagacacc 84000 tgcactcttg tttattacag cagtattcac tatagcaaag ataggaaatc aatccaagtg 84060 tgtatcaaca gataatagga taaagaaaat gtgatgtact cacacatata catacataca 84120 cacacacaca cacacacaca cacacacaaa atggaatagt attcagcaat caaaaagaat 84180 gaaagtgtgt cttttgcagc aacatggatg gaacaggaag tcattatctt aagtgaaaca 84240 attcagagac aaagtcagat accacatgtt ctcgcttata agtgggagct aaataatgca 84300 tgtacatggg catagagtat agaataattg acagtggtgc cttagaaggc tggaaaggta 84360 ggaggggggt gagacattac ctaatggata caatgtgcat tattgggtga tggttatacc 84420 aaaagctcag atttcaccac tatgcaatat attcatgtaa caaaactgca cagatggtca 84480 ttaaatttat acaaatttaa taaaaagagg ttctagatat acatatagat gtacatacta 84540 gagttaagtt ttcaattgac aaaaatttgc atagatatac tgaattgcca gaattactct 84600 tcagagtctc ctttatatac aagatcttga aaactcaaaa ccaaagaatt aacttatttg 84660 tttttcataa gtttgtatct ctggccttct tttctagggg aaatggaaac caagttttga 84720 ttggggtgca ggggataaat acaggcttaa gaaatagtaa ttttcctccc tgttccctga 84780 gatacaacaa tattggaatt aggccaaggc tgggcatggt ggctcaagcc cgtaattcca 84840 gcactttggg aagctgaggt gggcgaatca cttgaggtga ggagttcaag accagcctgg 84900 ccaatatagt gaaaccccat ctctaccaaa aagacaaaaa ttagtcggac atgatggcac 84960 acacttgtaa tcctagctac tcaggaggct gaggcgggag aattgcttga acatgggagg 85020 cggaggttgc agtgacctga gattgtgcca ctgaactcca gcctgggcga cagagcaaga 85080 ccctgtgtca aaaaaaaaaa aaaaaaaaaa aaagcaatta ggccaactaa caatcttgga 85140 atggcctctc agtgttccag taaaagaaag agttgcaagt ctgaggtaga aatgattaag 85200 cttaattagg aaggcatgtc aaaagttgat ataggttgaa aactaggtct cctgagccaa 85260 gcaactagcc aagtttcaaa tacaaagtaa gagttcttga aggactttaa aagtgccatt 85320 ccagtgaaca catgaatgat aagcaaaacc gccttattgc tgatatggag aaagttttag 85380 tggtctgaat agaagatcaa accagccaca atattccctt aagcctaagc ctcatccaga 85440 ggaaggccct gattctcctc aatactagga aggcaaagag atgtaaggaa ggagcagaaa 85500 aaagtttgaa gctagcagaa gttggttcat gagatttaag aagccatttc cataacaaag 85560 tataaggtga agcagcaagt gctgatgcag aagctgcagc aagttaccca gaagatctag 85620 gtaagattat cgctacttat gaaagtagct gtgctaaaaa acaggttttc agggtagata 85680 aaacagcttt ccactggaag aagatgtcat ctaggacttt catagctaca gagaacaagt 85740 caatgtctgt cttcaaagct tcaaatgaca ggttgactct cttgttaggg gctaatgaag 85800 ctggtgactt gaagctgaag ctggtgactt gaagctgaag ctaatgctca tttatcattc 85860 tgaagatcct aaggccttta agaattacgc tcaatctgtt ctgcctgtgg tttatgaatg 85920 gaacaacaaa gctggatgac agcacatcta tttatggcat ggtttactga atattttgac 85980 ccactattga gacttactgc tcagaaaaga atactacttt caaatattat tgctcattga 86040 caatgcacct agtcagccaa gagctcttat ggatatatac aaggagatta atgttttcat 86100 ttctgctaac acaacattca ttctgtagtt catagatcaa agagtaattt tccactttca 86160 tgtcttatta tttaagaaat atattttgta aagctgtggc tgccatagat agtgtttcct 86220 ctgatggatc tgggtaaagt aaattgaaaa ccttctagaa aggattcacc tttctagatg 86280 ccattaagaa cattcgtgat tcatgggaga aggtgaaaat agcaacatta acaagagttt 86340 ggaagaagtt tatttcatcc ctcttgcatg actttgaggg tgttcaaaac ttcagtggag 86400 gaaataacta cagatatgtg aaaagagcaa gagaactaaa attagaagta aagcctgaag 86460 acatgactga attgaagagg tttcgtggta aaacttgaaa gatgaggagt tgtttcttat 86520 ggatgagcaa agaaagtggt ttcttgagtt ggaatctact tctagtgaag atgctatgaa 86580 gattattgaa gtgacaacaa aggatttcgg agattacata aacttagttg ataatgcagc 86640 agcaggactt aagaggattg actccaatta tgaaagaagt tctattgtgg gtaaattgcc 86700 atcaaactgc tagcattgca tgctacagaa aaattattca caaaagagtc aactgatgca 86760 gcaaacttca ttgttgcctt attttgagaa actgtcatag ccaccccaac cttcagcaac 86820 caccaccctg gccaatcagc agccatcaac atttagacaa caccctccaa cagcaaaaag 86880 attttgactc agggaagact cagatgattg ttaccatttt ctagcaataa agtgtttttt 86940 agttaaggtg tgttcacttt ttaaataatg ctgttgcaca cttaatacat tctagtatag 87000 tgtaaatata acttctacgt ggactgggaa acaaaaagat gcatgtgatt cactttgttg 87060 caatatttcc ttcattgccg tggtctggaa ccaaaccggc tatatctcca agatatgcct 87120 gtgtaacaaa gtggctggat ataagataaa tatttaaata ttaatacctt tctcccatat 87180 cagcaggaag gcattagaaa acttaatgaa gcaattttta aaattacaac aaaaagataa 87240 atcatctagg aataatctta atggcaaata cttgagattt atataaagaa gacaaaagtt 87300 tactaagaga tccaagtatt cattaaaaag aagggaaaaa aaagatccag attacatgca 87360 gctgtaaact aagtcaaacc aagtatttaa gaagcatgta gaataaggcc aggttctctt 87420 ttgagcaaat gctaagttaa atgcacaatc cattgataca ggtctaaaat aggtcagctg 87480 aagtgttctt taatatacct attaattctt atgttcttcc agtgaactag aagtttatat 87540 gtcctgatat tgatagtgat atcaataaca ctatctcttt cctatataag gtgttttttt 87600 tctagagtca tgtatcagaa gaaatcatta tatagctcag aaatatatgt tataaattgt 87660 taatgtcatc tttttataca caactgccac ttttaattgt cttcttaaca gttcctttgg 87720 aagggctacg aagtcaaacc cagtttgagg agatgaggtc aagctacatt agagagctca 87780 tcaaggcaat tggtttgagg caaaaaggag ttgtgtcgag ctcacagcgt ttctatcaac 87840 ttacaaaact tcttgataac ttgcatgatg taagtatttg gttgattcca gaatatcaat 87900 gattattctc tgaatttcta taacttttta aatgttacat gtaaatttta ctttgtatga 87960 ttttctcaga ttaatacttg tatgttaaaa gtgtttggat catgctgctc agacttttta 88020 ttgtgttttt ttttcatttt caatatagac cactcaaata ttcttttaca agtatttgta 88080 taatgtgagg gtaatttaat agctgataga aattatggta catttcctga atatgttcta 88140 aaatatttgt gcctaaaatt ataaaagatt ttatatatac agtaaaaaca aaggtatgta 88200 tgtgaaaaat aagactcatg ccattttcag tggaattata aataacattg ctgatgggtg 88260 ggcaatcagg caatacatag caaaagttaa aatttgcatg cttttcaagt ttctattttt 88320 taggatttat cttaaagaaa taattagaca aatgtaatat acatgcaagg atgttcacca 88380 tagcagaggg ggaaaaaaga aaagcaaaca agaaatgccc aacaccagag gtggattact 88440 aagacaagat atctgtggtt tgtcaagata tatgaaatac aagcttttat tgctactatg 88500 ttcagatcac attaaaatga cagtgaaggc ataaaaaagc tatccactct ccaggtcaac 88560 ttttgaggtc ttactcctga atacgaacag acagccaaga ttatgagcta tttgagtaaa 88620 gcctctaaaa tgaaaggcag aaagcaaaat aaaaagaaac ttgaaggaaa taaacaccag 88680 tgcagagaac agaagaaaac ttctaacatc ctcagagaaa taagaatgat acggtatcca 88740 tgacatgaga acagaaaaca tttttaaaac agacatttag caagaaaata cttttggata 88800 attaaatgaa gaaaggcaga aattgaaaat tcaatgaaag ggctgaaatg gaaaggaaaa 88860 tctgaggaaa aataaaagat caaaacatgg aaaattagaa agaagagtta tagtaaattt 88920 agtattcagg agattctagc ctccaattaa taggaatgtc cagaagaaaa aatagagaaa 88980 atcaaggaga gaaatttgtg taagaaatcg caaaagttgc tggaactgaa acatacaagt 89040 tttctaacaa aaagggtccg gtgtagaggc actgcacaat tgaactaagg tacattattg 89100 taaaattgta ccaggccaga aatgaagaga atattttaaa gtataccagt gatgaaagaa 89160 acaggtcaca tagacgttat caggattcag aatgatgttg gacttctctg cagcaacact 89220 ggaagccaga agacagtgga gcagtgcctt cagaatttga tggaaaagaa aatcttcaaa 89280 gaatttcata cctagccaaa ccatcaaaca agtacaatgg cagcatgaaa gtattttcca 89340 acttgcaagg tctcctagag tttacctcag atacaacttt ctcaagaact gctacaggat 89400 gtcttataac aaaacatggg attaagccaa agatccaccc caacagagat gcaaagtgat 89460 gtcccagaac aataactgtg cagagcagta gttagtctag atgggagcag taatacaaag 89520 ggttctagga taccagctcc aggaaaaata aatggaactg agagattacc actaagtatg 89580 actgtaattg aggtgatttt tacaattctg ttgcagaatt tgactatggc tttgagacaa 89640 atatagagaa aacaaagcaa acaagaacac gaggtgattc acttttgcaa aataaaaata 89700 agtcgtaaga atggagatgc aatccagcag aaaaccataa aaaaatcttt atgatataaa 89760 tgctggatat tgactttagc taaaaattga gccaaaagct ctctctgggg agaagagaac 89820 atgtatgtag ttgaggaaga gagtgtctag tataagagag cttcattttc ttcatttcct 89880 atagtagaaa ctaaaactaa ctttagtgga aatatagaaa tatcaaaaga cacaggctac 89940 aaaaagttga aggtagttgt gtctagggag ctggaatagg gatgggaaga agtctatcag 90000 gaaactgcta ggctctattg atgctgctgc tgctgctgtt tttgttttta taatcatgtg 90060 gtactacttg actttcaaca ttatacacat gaattacttt gataaaaatt aaacgatgtt 90120 ggattattgg cataggaaga accaatatgt tgttatataa aacagctaga ttagcaaata 90180 gaatttatga aacatgtgta atattgtgtg tatgcttggg tatgtctgta tacatgtata 90240 aaaagaaaaa aaaacttgag taatacacca aaatgttcac acagcttatc ctgtagaatt 90300 gtgaataact ttcatttctt taaataaatc tttccataat atcagaatgt tctatcatga 90360 gtatataaca ttttacatat tattctagaa aatggtggga gttattttca ctatggtaag 90420 aaaaaaacac cttgagaagt atttatatta taaatagtta gaaaaataaa ttgccatgtt 90480 tgaatagcat atgaatttat tatttttatt acatgttttc tactcatttg ttaaaccaac 90540 agcttgtcaa acaacttcat ctgtactgct tgaatacatt tatccagtcc cgggcactga 90600 gtgttgaatt tccagaaatg atgtctgaag ttattgctgc acaattaccc aagatattgg 90660 cagggatggt gaaacccctt ctctttcata aaaagtgaat gtcatctttt tcttttaaag 90720 aattaaattt tgtggtatgt ctttttgttt tggtcaggat tatgaggtct tgagttttta 90780 taatgttctt ctgaaagcct tacatttata acatcatagt gtgtaaattt aaaagaaaaa 90840 ttgtgaggtt ctaattattt tcttttataa agtataatta gaatgtttaa ctgttttgtt 90900 tacccatatt ttcttgaaga atttacaaga ttgaaaaagt actaaaattg ttaaagtaaa 90960 ctatcttatc catattattt cataccatgt aggtgaggat ttttaacttt tgcatctaac 91020 aaatcatcga cttaagagaa aaaatcttac atgtaataac acaaagctat tatatgttat 91080 ttctaggtaa ctccctttgt gtcaattata tttccaaaaa tgaaccttta aaatggtatg 91140 caaaattttg tctatatata tttgtgtgag gaggaaattc ataactttcc tcagattttc 91200 aaaagtattt ttaatgcaaa aaatgtagaa agagtttaaa accactaaaa tagattgatg 91260 ttcttcaaac taggcaaaac aactcatatg ttaagaccat tttccagatt ggaaacacaa 91320 atctcttagg aagttaataa gtagattcat atcattatgc aaatagtatt gtgggttttg 91380 taggttttta aaataacctt ttttggggag agaattgtcc tctaatgagg tattgcgagt 91440 ggacataaga aatcagaaga ttatggccta actgtactcc ttaccaactg tggcatgctg 91500 aaagttagtc actcttactg attctcaatt ctctcacctt tgaaagtagt aaaatatctt 91560 tcctgccaat tgctcctttg ggtcagagct tattaacatc ttttcaaatc aaaggaaaga 91620 agaaagggag aggaggagga gggaggtatc aattcacata cctttctcct ctttatcctc 91680 cactatcatg aattcatatt atgtttcagc catgcaaatc tttttaccat gaaatttctt 91740 ccagaatttt ccccctttga cacaaattcc atgcatgttt caaccttcga gactcagcca 91800 aatgtcattt ctgtaaaatc ttccctgagt cttccaagca gtaatttgcc ttctcctaga 91860 gtttacctgc cattttgtgc acatttgagt tacagtagca tgttatttta caattgtgac 91920 tctcctggga gtctgggagc catataaagt ggtcaatagt gtttgctgac tgagagttga 91980 atgacatttt ctctctgtct tggtattact gtagatttcg atcattcttt ggttacattt 92040 ctgcatattt ctgtacccat gactttatca ctttcttctc ccatgcttta tctccatcaa 92100 ttatcttcat tacttttaaa ttttccacct ttgcttccta ctttgtgaga tctctccctt 92160 tactgactat aacatagaag aatagaagtg tattttatgt gtcttaagga caatacttta 92220 gattccttgt tctaagtttt taaactgaat gaatggaata ttatttctct ccctaagcaa 92280 aattccacaa aacaattatt tcttatgttt atgtagcctt aaattgtttt gtactgtaaa 92340 cctcagcata aaaactttct tcatttctaa tttcattcaa caaatattga ttgaatacct 92400 ggtattagca caagaaaaat gtgctaataa gccttatgag aatttggagc tgaagaaaga 92460 catataactc aggaaagtta cagtccagta gtaggtataa attacagtgc ctgataaata 92520 ggcattttaa tatttgtaca ctcaacgtat actaggtagg tgcaaaacat ttacatataa 92580 ttttactgat acccatgcag cacaaaggta ctaactttaa atattaaata acacctttat 92640 gtgtcagtaa ttcatttgca ttaaatctta ttgaaaaggc tttcaatata ttttccccac 92700 aaatgtcatc ccaagaaaaa agtattttta acatctccca aatataatag ttacaggaaa 92760 tctacctctg tgagagtgac acctctcaga atgaactgtg tgacacaaga aaatgaatgt 92820 aggtctatcc aaaaaaaacc ccaagaaaca aaaacaatat tattagccct ttatgcttaa 92880 gtgatggact cagggaacag ttgatgttgt gatcatttta ttatctgatt cttgttactt 92940 tgaattaaac caatattttg atgatataaa tcatttccac cagcatatat ttaatttcca 93000 taataacttt aaaattttct aatttcactc aactatgagg gaatagaatg tggtggccac 93060 aggtttggct tttgttaaaa tgtttgatat cttcgatgtt gatctctgtc tgcaatgtag 93120 atgtctaaac actaggattt aatatttaag gctaagcttt aaaaataaag taccttttta 93180 aaaagaatat ggcttcacca aatggaaaat acctaatttc taaatctttt tctctacaaa 93240 gtcctatcta ctaatgtctc cattactatt tagtcatcat aaccattatc ttcattttac 93300 atgtcgtgtt ctttctggta gctctaaaat gacactaaat cataagaaga caggttacat 93360 atcaggaaat acttgaaggt tactgaaata gattcttgag ttaatgaaaa tattttctgt 93420 aaaaaggttt gaaaagccat ttgagtctaa agcattatac ctccattatc agtagttatg 93480 tgacaattgt gtgtgtgttt aatgtttaaa gatgtggcac tttttaataa ggcaatgcta 93540 tgctattttt tcccatttaa cattaagata atttattgct atacagatga tatggaaata 93600 tgatgaacaa tatttttttt gccaaaacta tgccttgtaa gtagccatgg aatgtcaacc 93660 tgtaacttaa attatccaca gatagtcatg tgtttgatga tgggcactgt ggagataact 93720 gacataggac tgtgcccccc ttctctgcca cttactagct ggatgagatt aagcaagtca 93780 tttaactgct ctgattaaac ctgcctttcc caagtgcttt gtaatgaata gaaatggaaa 93840 ccaaaaaaaa cgtatacagg ccttcagaaa tagtaattgc tactattttg ttttcattaa 93900 gccatagttc tggctataat tttatcaaac tcaccagcta tattctacag tgaaagcagg 93960 attctagaaa gtctcactgt tttatttatg tcaccatgtg ctatgatata tttggttgaa 94020 ttcatttgaa attagggctg gaagtattca agtaatttct tctgctgaaa aaatacagtg 94080 ttttgagttt agggcctgtt ttatcaaagt tctaaagagc ctatcactct tccattgtag 94140 acattttaaa ataatgacac tgattttaac atttttaagt gtctttttag aacagagagc 94200 ctgactagaa cacagcccct ccaaaaaccc atgctcaaat tatttttact atggcagcaa 94260 ttccacaaaa gggaacaatg ggtttagaaa ttacaatgaa gtcatcaacc caaaaaacat 94320 ccctatccct aagaaggtta tgatataaaa tgcccacaag aaatctatgt ctgctttaat 94380 ctgtctttta ttgctttgga aggatggcta ttacattttt agtttttgct gtgaatacct 94440 gagcagtttc tctcatccat acttatcctt cacacatcag aagtcaggat agaatatgaa 94500 tcattttaaa aacttttaca actccagagc catgtgcata agaagcattc aaaacttgcc 94560 aaaacataca ttttttttca aatttaaaga tactctattt ttgtattcaa tagctcaaca 94620 actgtggtcc ccactgataa agtgaagtgg acaaggagac aagtaatggc ataagtttgt 94680 ttttcccaaa gtatgcctgt tcaatagcca ttggatgtgg gaaatttcta catctcttaa 94740 aattttacag aaaatacata gccagatagt ctagcaaaag ttcaccaagt cctaaattgc 94800 ttatccttac ttcactaagt catgaaatca ttttaatgaa aagaacatca cctaggtttt 94860 gtggtttctt tttttcttat tcatggctga gtgaaaacaa caatctctgt ttctccctag 94920 catctgtgga ctatttaatg taccattatt ccacactcta tggtccttac taaatacaaa 94980 attgaacaaa aagcagtaaa acaactgact cttcacccat attataaaat ataatccaag 95040 ccagattagt caacatccat aagatgaatc caagctgaac tgggcctaga ttattgagtt 95100 caggttggat cacatcccta tttattaata aacttaggaa agaaggcctt acagaccatc 95160 agttagctgg agctaataga acctacactt ctaaagttcg gcctagaatc aatgtggcct 95220 taaaagctga aaagaagcag gaaagaacag ttttcttcaa taatttgtcc accctgtcac 95280 tggagaaaat ttaagaattt gggggtgttg gtagtaagtt aaacacagca gctgttcatg 95340 gcagaaatta ttcaatacat accttctctg aatatcctat aaccaaagca aagaaaaaca 95400 ccaaggggtt tgttctcctc cttggagttg acctcattcc aaggcagagc tcaggtcaca 95460 ggcacagggg ctgcgcccaa gcttgtccgc agccttatgc agctgtggag tctggaagac 95520 tgttgcagga ctgctggcct agtcccagaa tgtcagcctc attttcgatt tactggctct 95580 tgttgctgta tgtcatgctg accttattgt taaacacagg tttgtttgct ttttttccac 95640 tcatggagac atgggagagg cattattttt aagctggttg aaagctttaa ccgataaagc 95700 atttttagag aaatgtgaat caggcagcta agaaagcata ctctgtccat tacggtaaag 95760 aaaatgcaca gattattaac tctgcagtgt ggcattagtg tcctggtcaa tattcggata 95820 gatatgaata aaatatttaa atggtattgt aaatagtttt caggacatat gctatagctt 95880 atttttatta tcttttgaaa ttgctcttaa tacatcaaat cctgatgtat tcaatttatc 95940 agatataaat tattctaaat gaagcccagt taaatgtttt tgtcttgtca gttatatgtt 96000 aagtttctga tctctttgtc tatgacgttt actaatctgc atttttactg ttatgaatta 96060 ttttagacag cagtggtttc aagctttttg ccactaaaaa taccttttat tttctcctcc 96120 cccagaaaag tctatacctt gaagtatcta tccaccaaac tgtacttcta ttaagaaata 96180 gttattgtgt tttcttaatg ttttgttatt caaagacata tcaatgaaag ctgctgagca 96240 gcatgaataa caattatatc cacacagatt tgatatattt tgtgcagcct taacttgata 96300 gtataaaatg tcattgcttt ttaaataata gttagtcaat ggacttctat catagctttc 96360 ctaaactagg ttaagatcca gagctttggg gtcataatat attacataca attaagttat 96420 ctttttctaa gggctttaaa attcatgaga ataaccaaaa aaggtatgtg gagagttaat 96480 acaaacatac catattcttg ttgaaacaga gatgtggctc tgcttgttct ccataaggta 96540 gaaatacttt ccagaatttg cctaaactag taagccctga atttgctatg attagggata 96600 ggaagagatt ttcacatggc agactttaga attcttcact ttagccagta aagtatctcc 96660 ttttgatctt agtattctgt gtattttaac ttttctgagt tgtgcatgtt tataagaaaa 96720 atcagcacaa agggtttaag ttaaagcctt tttactgaaa tttgaaagaa acagaagaaa 96780 atatcaaagt tctttgtatt ttgagaggat taaatatgat ttacaaaagt tacatggagg 96840 gctctctaaa acattaaatt aattattttt tgttgaaaag tcttacttta ggcatcattt 96900 tattcctcag caactagctg tgaagccttt actgtgctgt atgccagtca ctctgctaga 96960 ttgtggagat taccagtgtt cccgtcttct ccgagcttag agttggatgg ggaataaaga 97020 caggtaaaca gatagctaca atattgtact gtgaatgctt atgctggagg aagtacaggg 97080 aactattgga gcacctaaga ggagcaccta ccttgaattt aggggttagc agaggcatcc 97140 tgaaaaaagt caaagctaag ccacaatcta taagcagttt aggaattagc agaacgtgcg 97200 tggtgaggag atgccaaagg caagaagaga agagtattcc aaacaggagg gattccaaag 97260 agagaagagt atcccaaaca acatttgcac aaacctgatg gggagagaga atgtggggtg 97320 gggatggatg atgagactga agaagaaagc caggtctaga taatcagtgg ccttgtacac 97380 catgttaaag agtgtagact tgattctgtt gtaaacagga aagcagcaca attcatatga 97440 atattttaga agactcccac tggaatatgg agaataaagt tggagatgac taatcctgga 97500 agcagggaga acatttttga ggaagttgca ctattttggt gaaaatgatg atcataaaca 97560 tgaagaattg taggtgatca tgacctcctc tctaattttc cagaagggtt ttggaagata 97620 taacatagga acattgacag gactgacgaa aggagatgaa atacaccata taaattgtca 97680 aacacaaggc cagatgtcta attattttgc ttatgtgttg aaattacaaa tttttcatca 97740 ggaaaccaaa aactacaaaa cttagttttc ccaagtccca gaattctatc tgtccaaaca 97800 atctgtacca ctccacctat atccctacct ttgcatgtct gtccaacctc aaagtccagg 97860 tctatacaca cgggtaagac tagagcagtt caagtttcag aaaatgagaa agaggaactg 97920 agttgtgctg aacccataca aaataaacac attctttgta tagattcttg gaacctcgag 97980 aggaattcac ctaactcata ggtatttgat ggtatgaatc catggctggg ctcggctttt 98040 aaaaagcctt atctgggatt ccttctatgg aaccaagttc catcaaagcc catttaaaag 98100 cctacattaa aaacaaaatt cttgctgcat tgtatacaaa taatgatgtc atgatcaaat 98160 aatcagatgc cattatcaag tggaattaca aaatggtata cccactccaa aaaaaaaaaa 98220 aaagctaaat tctcagtaga acattgtgac ttcatgagcc ctccacagcc ttggagctga 98280 ggagggagca ctggtgagca gtaggttgaa gagaaaactt ggcgcttaat aatctatcca 98340 tgttttttca tctaaaagag ccttcttttt ggattacctt attcaatttc catcaaggaa 98400 attgttagtt ccactaacca gacagcagct gggaaggcag aagcttactg tatgtacatg 98460 gtagctgtgg gaaggaggtt tctttctcca ggtcctcact ggccatacac cagtcccttg 98520 ttagttatgc ctggtcatag acccccgttg ctatcatctc atatttaagt ctttggcttg 98580 tgaatttatc tattctttca gcttcagcac tgcagagtgc tgggactttg ctaacttcca 98640 tttcttgctg gcttagcaca ttcctcatag gcccagctct tttctca...

Examples

example 1

Cancer Patients Undergoing HER2 Targeted Therapy and Chemotherapy Benefit from PD-L1 Inhibitor Cotherapy, if the Expression Level of ER is Low or Absent (ER Negative) and if PD-L1 Expression Level is Increased

[0283]Estimation of gene expression was performed with the help of R Bioconductor package ‘affy’, R version 2.15.0. All exploratory analyses and predictive models were made using SAS JMP ver. 10.0

[0284]48 HER2+, ER+ and 39 HER2+, ER− breast cancer biopsies were obtained from NeoSphere clinical trial. The samples had been taken at diagnosis from patients afterwards treated with Docetaxel and Trastuzumab in a neo-adjuvant setting. The distribution of main clinical covariates at base line, as well as of clinical response (as assessed at the surgery) in the involved population is as follows:

ER Negative Samples:

Patient Age (see FIG. 17)Quantiles100.0%maximum72 99.5%72 97.5%71.55 90.0%64 75.0%quartile54 50.0%median50.5 25.0%quartile44.25 10.0%39 2.5%34.675 0.5%34 0.0%minimum34LevelCo...

Claims

1. A method of determining the need of a cancer patient for a PD-L1 inhibitor cotherapy, (i) wherein therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated for the patient or (ii) wherein the patient is undergoing therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent, the method comprising the steps ofa) measuring in vitro in a sample from the patient the expression level of Estrogen receptor (ER) and of programmed death ligand 1 (PD-L1),b) determining a patient as being in need of a PD-L1 inhibitor cotherapy if a low or absent ER expression level and an expression level of programmed death ligand 1 (PD-L1) that is increased in comparison to a control is measured in step (a).

2. A method of treating a cancer in a cancer patient for whom therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent is contemplated, the method comprising selecting a cancer patient whose cancer is determined to have a low or absent ER expression level and to have an increased expression level of programmed death ligand 1 (PD-L1) in comparison to a control, and administering to the patient an effective amount of a modulator of the HER2 / neu (ErbB2) signaling pathway, of a chemotherapeutic agent and of a programmed death ligand 1 (PD-L1) inhibitor.

3. A method of treating a cancer in a cancer patient who is undergoing therapy comprising a modulator of the HER2 / neu (ErbB2) signaling pathway and a chemotherapeutic agent, the method comprising selecting a cancer patient whose cancer is determined to have a low or absent ER expression level and to have an increased expression level of programmed death ligand 1 (PD-L1) in comparison to a control, and administering to the patient an effective amount of a programmed death ligand 1 (PD-L1) inhibitor.

4. The method of claim 1, further comprising measuring in vitro in a sample from the patient the expression level of interferon-gamma (IFNγ) and determining a patient as being in need of a PD-L1 inhibitor cotherapy if an expression level of interferon-gamma (IFNγ) that is decreased in comparison to a control is measured.

5. The method of claim, wherein the ER expression level is ER(−).

6. The method of claim 1, wherein the modulator of the HER2 / neu (ErbB2) signaling pathway is an inhibitor of HER shedding.

7. The method of claim 6, wherein the inhibitor of HER shedding is a HER2 shedding inhibitor.

8. The method of claim 6, wherein the inhibitor of HER shedding inhibits HER heterodimerization or HER homodimerization.

9. The method of claim 6, wherein the inhibitor of HER shedding is a HER antibody.

10. The method of claim 9, wherein the HER antibody binds to a HER receptor selected from the group consisting of EGFR, HER2 and HER3.

11. The method of claim 10, wherein the antibody binds to HER2.

12. The method of claim 11, wherein the HER2 antibody binds to sub-domain IV of the HER2 extracellular domain.

13. The method of claim 9, wherein the HER2 antibody is Herceptin / Trastuzumab.

14. The method of claim 1, wherein the modulator of the HER2 / neu (ErbB2) signaling pathway is a HER dimerization / signaling inhibitor.

15. The method of claim 14, wherein the HER dimerization inhibitor is a HER2 dimerization inhibitor.

16. The method of claim 14, wherein the HER dimerization inhibitor inhibits HER heterodimerization or HER homodimerization.

17. The method of claim 14, wherein the HER dimerization inhibitor is a anti HER antibody.

18. The method of claim 17, wherein the HER antibody binds to a HER receptor selected from the group consisting of EGFR, HER2 and HER3.

19. The method of claim 18, wherein the antibody binds to HER2.

20. The method of claim 19, wherein the anti HER2 antibody binds to domain II of HER2 extracellular domain.

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